Global distribution and genomic characteristics analysis of avian-derived mcr-1-positive Escherichia coli.

The prevalence of avian-derived Escherichia coli (E. coli) carrying mcr-1 poses a significant threat to the development of the poultry industry and public health safety. Despite ongoing in-depth epidemiological research worldwide, a comprehensive macroscopic study based on genomics is still lacking. In response, this study collected 1104 genomic sequences of avian-derived mcr-1-positive E. coli (MCRPEC) from the NCBI public database, covering 31 countries. The majority of sequences originated from China (48.82 %), followed by the Netherlands (10.41 %). In terms of avian hosts, chicken accounted for the largest proportion (44.11 %), followed by gallus (24.09 %). Avian-derived MCRPEC also serves as a reservoir for other antibiotic resistance genes (ARGs), with 179 ARGs coexisting with mcr-1 identified. A total of 206 virulence-associated genes were also identified, revealing the pathogenic risks of MCRPEC. Pan-genome analysis revealed that avian-derived MCRPEC from different hosts, countries of origin, and serotypes exhibit minor SNP differences, indicating a high risk of cross-regional and cross-host transmission. The ST types of MCRPRC are diverse, with ST10 being the most prevalent (n=70). Spearman analysis showed a significant correlation between the number of ARGs and the insertion sequences (ISs) as well as plasmid replicon in ST10 strains. Furthermore, ST10 strains share a similar genetic basis with human-derived MCRPEC, suggesting the possibility of clonal dissemination. Pan-genome-wide association studies (pan-GWAS) indicated that the differential genes of MCRPEC from different countries and host sources are significantly different, mainly related to genes encoding type IV secretion systems and mobile genetic elements (MGEs). Plasmid mapping of showed that the prevalent plasmid types vary by country and host, with IncI2 and IncX4 being the main mcr-1-positive plasmids. Among the 12 identified mcr-1 genetic contexts with ISs, the Tn6330 transposon was the predominant carrier of mcr-1. In summary, the potential threat of avian-derived MCRPEC cannot be ignored, and long-term and comprehensive monitoring are essential.

Functions of the UL51 protein during the herpesvirus life cycle.

The herpesvirus UL51 protein is a multifunctional tegument protein involved in the regulation of multiple aspects of the viral life cycle. This article reviews the biological characteristics of the UL51 protein and its functions in herpesviruses, including participating in the maintenance of the viral assembly complex (cVAC) during viral assembly, affecting the production of mature viral particles and promoting primary and secondary envelopment, as well as its positive impact on viral cell-to-cell spread (CCS) through interactions with multiple viral proteins and its key role in the proliferation and pathogenicity of the virus in the later stage of infection. This paper discusses how the UL51 protein participates in the life cycle of herpesviruses and provides new ideas for further research on UL51 protein function.

N-myc and STAT interactor degrades interferon regulatory factor 7 mediated type I interferon signaling to promote duck Tembusu virus replication.

N-myc and STAT interactor (NMI) is an interferon-induced protein, which plays a variety of biological functions by participating in signal transduction and transcriptional activation, it has been reported to regulate antiviral response of different viruses in many species. However, the role of NMI in ducks during Duck Tembusu Virus (DTMUV) infection is completely unknown. In order to reveal whether duck NMI (duNMI) is involved in the antiviral response in the process of DTMUV infection and its role, we cloned and identified duNMI gene, and conducted sequence analysis of duNMI, the open reading frame region of duNMI gene is 1,137 bp, encoding 378 amino acid residues (aa), including 3 domains, Coiled-coil domain (22-126aa), NMI/IFP 35 domain 1 (NID1) domain (174-261aa) and NMI/IFP 35 domain 2 (NID2) domain (272-360aa). Analysis of tissue distribution of duNMI in 7-day-old ducks shows that the expression of duNMI is the highest in harderian gland, followed by small intestine and pancreas. Subsequently, we found that mRNA level of duNMI increases significantly after DTMUV stimulation, and overexpression of duNMI inhibits DTMUV replication in a dose-dependent manner. Besides, duNMI inhibits the transcriptional activity of IFN-I related cytokines. Specifically, we confirmed that duNMI interacts with duck regulatory factor 7 (duIRF7) through NID1 and NID2 domains and inhibit its expression and activated-IFN-ß. These results support that duNMI is an inhibitor of antiviral innate immune response in the process of DTMUV infection, which will provide a theoretical basis for the prevention of DTMUV infection.

Vector competence of Culex quinquefasciatus for Tembusu virus and viral factors for virus transmission by mosquitoes.

The ongoing epidemic of flaviviruses worldwide has underscored the importance of studying flavivirus vector competence, considering their close association with mosquito vectors. Tembusu virus is an avian-related mosquito-borne flavivirus that has been an epidemic in China and Southeast Asia since 2010. However, the reason for the outbreak of Tembusu virus in 2010 remains unclear, and it is unknown whether changes in vector transmission played an essential role in this process. To address these questions, we conducted a study using Culex quinquefasciatus as a model for Tembusu virus infection, employing both oral infection and microinjection methods. Our findings confirmed that both vertical and venereal transmission collectively contribute to the cycle of Tembusu virus within the mosquito population, with persistent infections observed. Importantly, our data revealed that the prototypical Tembusu virus MM_1775 strain exhibited significantly greater infectivity and transmission rates in mosquitoes than did the duck Tembusu virus (CQW1 strain). Furthermore, we revealed that the viral E protein and 3' untranslated region are key elements responsible for these differences. In conclusion, our study sheds light on mosquito transmission of Tembusu virus and provides valuable insights into the factors influencing its infectivity and transmission rates. These findings contribute to a better understanding of Tembusu virus epidemiology and can potentially aid in the development of strategies to control its spread.

Duck enteritis virus LORF4 gene is a late gene and nonessential for virus replication in vitro.

Duck enteritis virus (DEV) is an avian alpha-herpesvirus that primarily causes an acute and highly contagious infectious disease of ducks. The LORF4 gene is one of the specific genes of DEV, with limited reports on its biological characteristics and functions. This study investigates the basic biological properties of LORF4 protein (pLORF4). The results show that DEV LORF4 is a late gene mainly localized in the cytoplasm of DEV-infected DEF. To explore the role of pLORF4 in the DEV replication life cycle, a recombinant virus lacking pLORF4 expression was constructed. The results showed that pLORF4 is not essential for virus replication and does not affect virus adsorption, assembly and release, it plays a positive role in virus invasion and DNA replication. In summary, this study provides a foundation for further research on the function of the LORF4 gene.

Duck hepatitis A virus utilizes PCBP2 to facilitate viral translation and replication.

Duck hepatitis A virus type 1 (DHAV-1) is an important member of the Picornaviridae family that causes highly fatal hepatitis in ducklings. Since picornaviruses have small genomes with limited coding capacity, they must utilize host proteins for viral cap-independent translation and RNA replication. Here, we report the role of duck poly(rC)-binding protein 2 (PCBP2) in regulating the replication and translation of DHAV-1. During DHAV-1 infection, PCBP2 expression was upregulated. A biotinylated RNA pull-down assay revealed that PCBP2 positively regulates DHAV-1 translation through specific interactions with structural domains II and III of the DHAV-1 internal ribosome entry site (IRES). Further studies revealed that PCBP2 promotes DHAV-1 replication via an interaction of its KH1 domain (aa 1-92) with DHAV-1 3Dpol. Thus, our studies demonstrated the specific role of PCBP2 in regulating DHAV-1 translation and replication, revealing a novel mechanism by which host‒virus interactions regulate viral translation and replication. These findings contribute to further understanding of the pathogenesis of picornavirus infections.

The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation.

Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The greasy finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only 5 rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.

Duck hepatitis A virus 1-encoded 2B protein disturbs ion and organelle homeostasis to promote NF-κB/NLRP3-mediated inflammatory response.

Previous studies by our group and others have highlighted the critical role of hyperinflammation in the pathogenicity of duck hepatitis A virus 1 (DHAV-1), an avian picornavirus that has caused significant devastation in the duck industry worldwide for decades. However, the precise mechanisms by which DHAV-1 infection regulates the inflammatory responses, particularly the production of IL-1ß, remain poorly understood. In this study, we demonstrate that DHAV-1 infection triggers NF-κB- and NLRP3 inflammasome-mediated IL-1ß production. Mechanistically, DHAV-1 infection, particularly its replication and translation, disrupts cellular homeostasis of Ca2+, K+, ROS and cathepsin, which act cooperatively as assembly signals for NLRP3 inflammasome activation. By screening DHAV-1-encoded proteins, we identified that the viroporin 2B dominates NF-κB as well as NLRP3 inflammasome activation. Mutation analysis revealed that I43 within the 2B protein is the key amino acid for Ca2+ mobilization and subsequent activation of NF-κB transcriptional activity and NLRP3 inflammasome. Moreover, DHAV-1 infection and the 2B protein activate the MAVS- and MyD88-NF-κB pathways by relay, providing the necessary priming signals for NLRP3 inflammasome activation. In summary, our findings elucidate a mechanism through which DHAV-1 triggers inflammatory responses via NF-κB/NLRP3 inflammasome activation, offering new perspectives on DHAV-1 pathogenesis and informing the development of targeted anti-DHAV-1 treatments.

Host miRNA and mRNA profiles during in DEF and duck after DHAV-1 infection.

DHAV-1 is a highly infectious pathogen that can cause acute hepatitis in ducklings. MicroRNA (miRNA) plays an essential regulatory role in virus response. We characterized and compared miRNA and mRNA expression profiles in duck embryonic fibroblasts (DEF) and the liver of ducklings infected with DHAV-1. DHAV-1 infected DEF was divided into infection group (D group) and blank group (M group), and DHAV-1 infected duckling group was divided into infection group (H group) and blank group (N group). D vs. M have 130 differentially expressed (DE) miRNA (DEM) and 2204 differentially expressed (DE) mRNA (DEG), H vs. N have 72 DEM and 1976 DEG. By the intersection of D vs. M and H vs. N comparisons, 15 upregulated DEM, 5 downregulated DEM, 340 upregulated DEG and 50 downregulated DEG were found with both in vivo and in vitro DHAV-1 infection. In particular, we identified the same DE miRNA target genes and functional annotations of DE mRNA. We enriched with multiple gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, which may have important roles in viral virulence, host immunity, and metabolism. We selected miR-155, which is co-upregulated, and found that miR-155 targets SOCS1 to inhibit DHVA-1 replication.

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.

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.

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.

Myricetin inhibits transmissible gastroenteritis virus replication by targeting papain-like protease deubiquitinating enzyme activity.

Myricetin, a natural flavonoid found in various foods, was investigated for its antiviral effect against transmissible gastroenteritis virus (TGEV). This α-coronavirus causes significant economic losses in the global swine industry. The study focused on the papain-like protease (PLpro), which plays a crucial role in coronavirus immune evasion by mediating deubiquitination. Targeting PLpro could potentially disrupt viral replication and enhance antiviral responses. The results demonstrated that myricetin effectively inhibited TGEV-induced cytopathic effects in a dose-dependent manner, with an EC50 value of 31.19 µM. Myricetin significantly reduced TGEV viral load within 48 h after an 8-h co-incubation period. Further investigations revealed that myricetin at a concentration of 100 µM directly inactivated TGEV and suppressed its intracellular replication stage. Moreover, pretreatment with 100 µM myricetin conferred a protective effect on PK-15 cells against TGEV infection. Myricetin competitively inhibited PLpro with an IC50 value of 6.563 µM. Molecular docking experiments show that myricetin binds to the Cys102 residue of PLpro through conventional hydrogen bonds, Pi-sulfur, and Pi-alkyl interactions. This binding was confirmed through site-directed mutagenesis experiments, indicating myricetin as a potential candidate for preventing and treating TGEV infection.

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.

Gallnut tannic acid alleviates gut damage induced by Salmonella pullorum in broilers by enhancing barrier function and modulating microbiota.

Pullorum disease (PD) is a bacterial infection caused by Salmonella pullorum (S. pullorum) that affects poultry. It is highly infectious and often fatal. Antibiotics are currently the mainstay of prophylactic and therapeutic treatments for PD, but their use can lead to the development of resistance in pathogenic bacteria and disruption of the host's intestinal flora. We added neomycin sulfate and different doses of tannic acid (TA) to the drinking water of chicks at 3 days of age and infected them with PD by intraperitoneal injection of S. pullorum at 9 days of age. We analyzed intestinal histopathological changes and the expression of immune-related genes and proteins by using the plate smear method, histological staining, real-time fluorescence quantitative PCR, ELISA kits, and 16S rRNA Analysis of intestinal flora. The results demonstrate that S. pullorum induces alterations in the immune status and impairs the functionality of the liver and intestinal barrier. We found that tannic acid significantly ameliorated S. pullorum-induced liver and intestinal damage, protected the intestinal physical and chemical barriers, restored the intestinal immune barrier function, and regulated the intestinal flora. Our results showed that TA has good anti-diarrhoeal, growth-promoting, immune-regulating, intestinal barrier-protecting and intestinal flora-balancing effects, and the best effect was achieved at an additive dose of 0.2%.

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.

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.

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.

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.