The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation.

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.

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.

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.

Citrullination profile analysis reveals peptidylarginine deaminase 3 as an HSV-1 target to dampen the activity of candidate antiviral restriction factors.

Herpes simplex virus 1 (HSV-1) is a neurotropic virus that remains latent in neuronal cell bodies but reactivates throughout an individual's life, causing severe adverse reactions, such as herpes simplex encephalitis (HSE). Recently, it has also been implicated in the etiology of Alzheimer's disease (AD). The absence of an effective vaccine and the emergence of numerous drug-resistant variants have called for the development of new antiviral agents that can tackle HSV-1 infection. Host-targeting antivirals (HTAs) have recently emerged as promising antiviral compounds that act on host-cell factors essential for viral replication. Here we show that a new class of HTAs targeting peptidylarginine deiminases (PADs), a family of calcium-dependent enzymes catalyzing protein citrullination, exhibits a marked inhibitory activity against HSV-1. Furthermore, we show that HSV-1 infection leads to enhanced protein citrullination through transcriptional activation of three PAD isoforms: PAD2, PAD3, and PAD4. Interestingly, PAD3-depletion by specific drugs or siRNAs dramatically inhibits HSV-1 replication. Finally, an analysis of the citrullinome reveals significant changes in the deimination levels of both cellular and viral proteins, with the interferon (IFN)-inducible proteins IFIT1 and IFIT2 being among the most heavily deiminated ones. As genetic depletion of IFIT1 and IFIT2 strongly enhances HSV-1 growth, we propose that viral-induced citrullination of IFIT1 and 2 is a highly efficient HSV-1 evasion mechanism from host antiviral resistance. Overall, our findings point to a crucial role of citrullination in subverting cellular responses to viral infection and demonstrate that PAD inhibitors efficiently suppress HSV-1 infection in vitro, which may provide the rationale for their repurposing as HSV-1 antiviral drugs.

Genome-wide association study reveals serovar-associated genetic loci in Riemerella anatipestifer.

BACKGROUND: The disease caused by Riemerella anatipestifer (R. anatipestifer, RA) results in large economic losses to the global duck industry every year. Serovar-related genomic variation, such as the O-antigen and capsular polysaccharide (CPS) gene clusters, has been widely used for serotyping in many gram-negative bacteria. RA has been classified into at least 21 serovars based on slide agglutination, but the molecular basis of serotyping is unknown. In this study, we performed a pan-genome-wide association study (Pan-GWAS) to identify the genetic loci associated with RA serovars. RESULTS: The results revealed a significant association between the putative CPS synthesis gene locus and the serological phenotype. Further characterization of the CPS gene clusters in 11 representative serovar strains indicated that they were highly diverse and serovar-specific. The CPS gene cluster contained the key genes wzx and wzy, which are involved in the Wzx/Wzy-dependent pathway of CPS synthesis. Similar CPS loci have been found in some other species within the family Weeksellaceae. We have also shown that deletion of the wzy gene in RA results in capsular defects and cross-agglutination. CONCLUSIONS: This study indicates that the CPS synthesis gene cluster of R. anatipestifer is a serotype-specific genetic locus. Importantly, our finding provides a new perspective for the systematic analysis of the genetic basis of the R anatipestifer serovars and a potential target for establishing a complete molecular serotyping scheme.

Prognostic impact of HER2 biomarker levels in trastuzumab-treated early HER2-positive breast cancer.

BACKGROUND: Overexpression of human epidermal growth factor receptor 2 (HER2) caused by HER2 gene amplification is a driver in breast cancer tumorigenesis. We aimed to investigate the prognostic significance of manual scoring and digital image analysis (DIA) algorithm assessment of HER2 copy numbers and HER2/CEP17 ratios, along with ERBB2 mRNA levels among early-stage HER2-positive breast cancer patients treated with trastuzumab. METHODS: This retrospective study comprised 371 early HER2-positive breast cancer patients treated with adjuvant trastuzumab, with HER2 re-testing performed on whole tumor sections. Digitized tumor tissue slides were manually scored and assessed with uPath HER2 Dual ISH image analysis, breast algorithm. Targeted ERBB2 mRNA levels were assessed by the Xpert® Breast Cancer STRAT4 Assay. HER2 copy number and HER2/CEP17 ratio from in situ hybridization assessment, along with ERBB2 mRNA levels, were explored in relation to recurrence-free survival (RFS). RESULTS: The analysis showed that patients with tumors with the highest and lowest manually counted HER2 copy number levels had worse RFS than those with intermediate levels (HR = 2.7, CI 1.4-5.3, p = 0.003 and HR = 2.1, CI 1.1-3.9, p = 0.03, respectively). A similar trend was observed for HER2/CEP17 ratio, and the DIA algorithm confirmed the results. Moreover, patients with tumors with the highest and the lowest values of ERBB2 mRNA had a significantly worse prognosis (HR = 2.7, CI 1.4-5.1, p = 0.003 and HR = 2.8, CI 1.4-5.5, p = 0.004, respectively) compared to those with intermediate levels. CONCLUSIONS: Our findings suggest that the association between any of the three HER2 biomarkers and RFS was nonlinear. Patients with tumors with the highest levels of HER2 gene amplification or ERBB2 mRNA were associated with a worse prognosis than those with intermediate levels, which is of importance to investigate in future clinical trials studying HER2-targeted therapy.

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.

Duck Tembusu virus NS3 protein induces apoptosis by activating the PERK/PKR pathway and mitochondrial pathway.

IMPORTANCE: Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that replicates well in mosquito, bird, and mammalian cells. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in the serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and poses a threat to mammalian health. Thus, understanding the pathogenic mechanism of DTMUV is crucial for identifying potential antiviral targets. In this study, we discovered that NS3 can induce the mitochondria-mediated apoptotic pathway through the PERK/PKR pathway; it can also interact with voltage-dependent anion channel 2 to induce apoptosis. Our findings provide a theoretical basis for understanding the pathogenic mechanism of DTMUV infection and identifying potential antiviral targets and may also serve as a reference for exploring the pathogenesis of other flaviviruses.

Characterization of a Unique Novel LORF3 Protein of Duck Plague Virus and Its Potential Pathogenesis.

Duck plague virus (DPV) is a high-morbidity fowl alphaherpesvirus that causes septicemic lesions in various organs. Most DPV genes are conserved among herpesviruses, while a few are specific to fowl herpesviruses, including the LORF3 gene, for which there is currently no literature describing its biological properties and functions. This study first addressed whether the LORF3 protein is expressed by making specific polyclonal antibodies. We could demonstrate that DPV LORF3 is an early gene and encodes a protein involved in virion assembly, mainly localized in the nucleus of DPV-infected DEF cells. To investigate the role of this novel LORF3 protein in DPV pathogenesis, we generated a recombinant virus that lacks expression of the LORF3 protein. Our data revealed that the LORF3 protein is not essential for viral replication but contributes to DPV replication in vitro and in vivo and promotes duck plague disease morbidity and mortality. Interestingly, deletion of the LORF3 protein abolished thymus atrophy in DPV-vaccinated ducks. In conclusion, this study revealed the expression of avian herpesviruses-specific genes and unraveled the role of the early protein LORF3 in the pathogenesis of DPV. IMPORTANCE DPV is a highly lethal alphaherpesvirus that causes duck plague in birds of the order Anseriformes. The virus has caused huge economic losses to the poultry industry due to high morbidity and mortality and the cost of vaccination. DPV encodes 78 open reading frames (ORFs), and these genes are involved in various processes of the viral life cycle. Functional characterization of DPV genes is important for understanding the complex viral life cycle and DPV pathogenesis. Here, we identified a novel protein encoded by LORF3, and our data suggest that the LORF3 protein is involved in the occurrence and development of duck plague.

RNF123 Mediates Ubiquitination and Degradation of SOCS1 To Regulate Type I Interferon Production during Duck Tembusu Virus Infection.

Many RING domain E3 ubiquitin ligases play critical roles in fine-tuning the innate immune response, yet little is known about their regulatory role in flavivirus-induced innate immunity. In previous studies, we found that the suppressor of cytokine signaling 1 (SOCS1) protein mainly undergoes lysine 48 (K48)-linked ubiquitination. However, the E3 ubiquitin ligase that promotes the K48-linked ubiquitination of SOCS1 is unknown. In the present study, we found that RING finger protein 123 (RNF123) binds to the SH2 domain of SOCS1 through its RING domain and facilitates the K48-linked ubiquitination of the K114 and K137 residues of SOCS1. Further studies found that RNF123 promoted the proteasomal degradation of SOCS1 and promoted Toll-like receptor 3 (TLR3)- and interferon (IFN) regulatory factor 7 (IRF7)-mediated type I IFN production during duck Tembusu virus (DTMUV) infection through SOCS1, ultimately inhibiting DTMUV replication. Overall, these findings demonstrate a novel mechanism by which RNF123 regulates type I IFN signaling during DTMUV infection by targeting SOCS1 degradation. IMPORTANCE In recent years, posttranslational modification (PTM) has gradually become a research hot spot in the field of innate immunity regulation, and ubiquitination is one of the critical PTMs. DTMUV has seriously endangered the development of the waterfowl industry in Southeast Asian countries since its outbreak in 2009. Previous studies have shown that SOCS1 is modified by K48-linked ubiquitination during DTMUV infection, but E3 ubiquitin ligase catalyzing the ubiquitination of SOCS1 has not been reported. Here, we identify for the first time that RNF123 acts as an E3 ubiquitin ligase that regulates TLR3- and IRF7-induced type I IFN signaling during DTMUV infection by targeting the K48-linked ubiquitination of the K114 and K137 residues of SOCS1 and the proteasomal degradation of SOCS1.

Functional characterization of a TerC family protein of Riemerella anatipestifer in manganese detoxification and virulence.

Manganese (Mn) is an essential element for bacteria, but the overload of manganese is toxic. In a previous study, we showed that the cation diffusion facilitator protein MetA and the resistance-nodulation-division efflux pump MetB are responsible for Mn efflux in the bacterial pathogen Riemerella anatipestifer CH-1. However, whether this bacterium encodes additional manganese efflux proteins is unclear. In this study, we show that R. anatipestifer CH-1 encodes a tellurium resistance C (TerC) family protein with low similarity to other characterized TerC family proteins. Compared to the wild type (WT), the terC mutant of R. anatipestifer CH-1 (∆terC) is sensitive to Mn(II) intoxication. The ability of TerC to export manganese is higher than that of MetB but lower than that of MetA. Consistently, terC deletion (∆terC) led to intracellular accumulation of Mn2+ under excess manganese conditions. Further study showed that ∆terC was more sensitive than the WT to the oxidant hypoclorite but not to hydrogen peroxide. Mutagenesis studies showed that the mutant at amino acid sites of Glu116 (E116), Asp122 (D122), Glu245 (E245) Asp248 (D248), and Asp254 (D254) may be involved in the ability of TerC to export manganese. The transcription of terC was upregulated under excess manganese and downregulated under iron-limited conditions. However, this was not dependent on the manganese metabolism regulator MetR. In contrast to a strain lacking the manganese efflux pump MetA or MetB, the terC mutant is attenuated in virulence in a duckling model of infection due to increased sensitivity to duck serum. Finally, comparative analysis showed that homologs of TerC are distributed across the bacterial kingdom, suggesting that TerC exerts a conserved manganese efflux function.IMPORTANCERiemerella anatipestifer is a notorious bacterial pathogen of ducks and other birds. In R. anatipestifer, the genes involved in manganese efflux have not been completely identified, although MetA and MetB have been identified as two manganese exporters. Additionally, the function of TerC family proteins in manganese efflux is controversial. Here, we demonstrated that a TerC family protein helps prevent Mn(II) intoxication in R. anatipestifer and that the ability of TerC to export manganese is intermediate compared to that of MetA and MetB. Sequence analysis and mutagenesis studies showed that the conserved key amino sites of TerC are Glu116, Asp122, Glu245, Asp248, and Asp254. The transcription of terC was regulated by manganese excess and iron limitation. Finally, we show that TerC plays a role in the virulence of R. anatipestifer due to the increased sensitivity to duck serum, rather than the increased sensitivity to manganese. Taken together, these results expand our understanding of manganese efflux and the pathogenic mechanisms of R. anatipestifer.

Differential pathogenic and molecular features in neurological infection of SARS-CoV-2 Omicron BA.5.2 and BA.2.75 and Delta.

The Coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a global threat, exacerbated by the emergence of viral variants. Two variants of SARS-CoV-2, Omicron BA.2.75 and BA.5, led to global infection peaks between May 2022 and May 2023, yet their precise characteristics in pathogenesis are not well understood. In this study, we compared these two Omicron sublineages with the previously dominant Delta variant using a human angiotensin-converting enzyme 2 knock-in mouse model. As expected, Delta exhibited higher viral replication in the lung and brain than both Omicron sublineages which induced less severe lung damage and immune activation. In contrast, the Omicron variants especially BA.5.2 showed a propensity for cellular proliferation and developmental pathways. Both Delta and BA.5.2 variants, but not BA.2.75, led to decreased pulmonary lymphocytes, indicating differential adaptive immune response. Neuroinvasiveness was shared with all strains, accompanied by vascular abnormalities, synaptic injury, and loss of astrocytes. However, Immunostaining assays and transcriptomic analysis showed that BA.5.2 displayed stronger immune suppression and neurodegeneration, while BA.2.75 exhibited more similar characteristics to Delta in the cortex. Such differentially infectious features could be partially attributed to the weakened interaction between Omicron Spike protein and host proteomes decoded via co-immunoprecipitation followed by mass spectrometry in neuronal cells. Our present study supports attenuated replication and pathogenicity of Omicron variants but also highlights their newly infectious characteristics in the lung and brain, especially with BA.5.2 demonstrating enhanced immune evasion and neural damage that could exacerbate neurological sequelae.

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 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.

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.

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.

Characteristics of the a sequence of the duck Plague virus genome and specific cleavage of the viral genome based on the a sequence.

During the replication process, the herpesvirus genome forms the head-to-tail linked concatemeric genome, which is then cleaved and packaged into the capsid. The cleavage and packing process is carried out by the terminase complex, which specifically recognizes and cleaves the concatemeric genome. This process is governed by a cis-acting sequence in the genome, named the a sequence. The a sequence and genome cleavage have been described in some herpesviruses, but it remains unclear in duck plague virus. In this study, we analysed the location, composition, and conservation of a sequence in the duck plague virus genome. The structure of the DPV genome has an a sequence of (DR4)m-(DR2)n-pac1-S termini (32 bp)-L termini (32 bp)-pac2, and the length is 841 bp. Direct repeat (DR) sequences are conserved in different DPV strains, but the number of DR copies is inconsistent. Additionally, the typical DR1 sequence was not found in the DPV a sequence. The Pac1 and pac2 motifs are relatively conserved between DPV and other herpesviruses. Cleavage of the DPV concatemeric genome was detected, and the results showed that the DPV genome can form a concatemer and is cleaved into a monomer at a specific site. We also established a sensitive method, TaqMan dual qRT‒PCR, to analyse genome cleavage. The ratio of concatemer to total viral genome was decreased during the replication process. These results will be critical for understanding the process of DPV genome cleavage, and the application of TaqMan dual qRT‒PCR will greatly facilitate more in-depth research.

N-glycosylation of the envelope glycoprotein I is essential for the proliferation and virulence of the duck plague virus.

Duck plague virus (DPV) causes the highly pathogenic duck plague, and the envelope glycoprotein I (gI), as one of the key virulence genes, has not yet had its critical virulence sites identified through screening. This study used reverse genetics technology to target the gI, specifically within the DPV genome. Four DPV mutants with gI N-glycosylation site mutations were designed and constructed, and these mutant strains were successfully rescued. Our results confirmed that three asparagine residues of gI (N69, N78, and N265) are N-glycosylation sites, and western blot analysis substantiated that glycosylation at each predicted N-glycosylation site was compromised. The deglycosylation of gI leads to the protein misfolding and subsequent retention in the endoplasmic reticulum (ER). The subsequent deglycosylated gI is carried into the Golgi apparatus (GM130) in the interaction of gE. Compared to the parental virus, the mutated virus shows a 66.3% reduction in intercellular transmission capability. In ducks, the deglycosylation of gI significantly reduces DPV replication in vivo, thereby weakening the virulence of DPV. This study represents the first successful creation of a weak DPV virus strain by specific mutation at the N-glycosylation site. The findings provide a foundational understanding of DPV pathogenesis and form the basis for developing live attenuated vaccines against the disease.

Dynamics of sediment-associated nitrogen export from intermittent streams in subtropical forests of southeast China.

The significance of intermittent streams in nutrient loss within forest ecosystems is becoming increasingly critical due to changes in precipitation patterns associated with global climate change. However, few studies have focused on nutrient export from intermittent streams. We conducted continuous sediment collection from intermittent streams from March 2022 to February 2023 to investigate the export pattern and mechanism of sediment-associated nitrogen (N) from intermittent streams of different forest types (composed forest of Castanopsis carlesii (Cas. carlesii) and Cunninghamia lanceolata (C. lanceolata) forests, compared to Cas. carlesii forests). We measured the N concentrations and calculated the export amounts of four common forms of N associated with sediments: total N (TN), dissolved N (DN), nitrate, and ammonia. Our results showed that (1) the annual average exports of TN, DN, nitrate, and ammonia associated with sediments from intermittent streams from both forest types were 273, 1.62, 0.26, and 0.84 kg ha-1, respectively; (2) N export was significantly higher in composite forests of Cas. carlesii and C. lanceolata, compared to Cas. carlesii forests; (3) stream sediment export amount positively affected N export both in composite forests and Cas. carlesii forests; and (4) N export was also controlled by rainfall amount and stream characteristics. Our study quantified sediment-associated N export from intermittent streams among different subtropical forest types, which will enhance our understanding of N dynamics associated with stream hydrological processes in subtropical forests.

Percutaneous microwave ablation versus sclerotherapy for large hepatic hemangioma: a multi-center cohort study.

OBJECTIVE: The study aimed to compare the effectiveness and safety of ultrasound-guided microwave ablation (MWA) and percutaneous sclerotherapy (PS) for the treatment of large hepatic hemangioma (LHH). METHODS: This retrospective study included 96 patients who underwent MWA (n = 54) and PS (n = 42) as first-line treatment for LHH in three tertiary hospitals from January 2016 to December 2021. Primary outcomes were technique efficacy rate (volume reduction rate [VRR] > 50% at 12 months), symptom relief rate at 12 months and local tumor progression (LTP). Secondary outcomes included procedure time, major complications, treatment sessions, cost and one-, two-, three-year VRR. RESULTS: During a median follow-up of 36 months, the MWA group showed a higher technique efficacy rate (100% vs. 90.4%, p = .018) and symptom relief rate (100% vs. 80%, p = .123) than the PS group. The MWA group had fewer treatment sessions, higher one-, two- and three-year VRR, lower LTP rate (all p < .05), longer procedure time and higher treatment costs than the PS group (both p < .001). MWA shared a comparable major complications rate (1.8% vs. 2.4%, p = .432) with PS. After multivariate analysis, the lesion's heterogeneity and maximum diameter >8.1 cm were independent risk factors for LTP (all p < .05). In the PS group, lesions with a cumulative dose of bleomycin > 0.115 mg/cm3 had a lower risk of LTP (p = .006). CONCLUSIONS: Both MWA and PS treatments for large hepatic hemangioma are safe and effective, with MWA being superior in terms of efficacy.