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BackgroundDuck enteritis virus (DEV) belongs to Alphaherpesvirinae; little is known about the DEV UL7 gene and its encoded protein. This study examined the molecular characteristics of DEV pUL7 in vitro and determined whether DEV pUL7 co-localizes with pUL51.ResultsThe results showed that UL7 can be regarded as a late gene. Moreover, immunofluorescence assay revealed that pUL7 was located around the perinuclear cytoplasmic region and co-localized with pUL51 in the cytoplasm and nucleus after transfection into duck embryo fibroblast cells (DEFs).ConclusionIn conclusion, we identified the molecular characteristics of the DEV UL7 gene, which is a late gene, and the co-localization of its encoded protein with pUL51 in transfected DEFs, enriching our understanding of pUL7 and future research directions.

Abstract 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.

Abstract 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.

ABSTRACT: 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.

ABSTRACT: 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.

ABSTRACTRiemerella anatipestifer (R. anatipestifer) is an important pathogen that causes severe systemic infections in domestic ducks, resulting in substantial economic losses for China’s waterfowl industry. Controlling R. anatipestifer with antibiotics is extremely challenging due to its multidrug resistance. Notably, large-scale studies on antimicrobial resistance (AMR) and the corresponding genetic determinants in R. anatipestifer remain scarce. To solve this dilemma, more than 400 nonredundant R. anatipestifer isolates collected from 22 provinces in China between 1994 and 2021 were subjected to broth dilution antibiotic susceptibility assays, and their resistance-associated genetic determinants were characterized by whole-genome sequencing. While over 90% of the isolates was resistant to sulfamethoxazole, kanamycin, gentamicin, ofloxacin, norfloxacin, and trimethoprim, 88.48% of the isolates was resistant to the last-resort drug (tigecycline). Notably, R. anatipestifer resistance to oxacillin, norfloxacin, ofloxacin, and tetracycline was found to increase relatively over time. Genome-wide analysis revealed the alarmingly high prevalence of blaOXA-like (93.05%) and tet(X) (90.64%) genes and the uneven distribution of resistance genes among lineages. Overall, this study reveals a serious AMR situation regarding R. anatipestifer in China, with a high prevalence and high diversity of antimicrobial resistance genes, providing important data for the rational use of antibiotics in veterinary practice.IMPORTANCERiemerella anatipestifer (R. anatipestifer), an important waterfowl pathogen, has caused substantial economic losses worldwide, especially in China. Antimicrobial resistance (AMR) is a major challenge in controlling this pathogen. Although a few studies have reported antimicrobial resistance in R. anatipestifer, comprehensive data remain a gap. This study aims to address the lack of information on R. anatipestifer AMR and its genetic basis. By analyzing more than 400 isolates collected over two decades, this study reveals alarming levels of resistance to several antibiotics, including drugs of last resort. The study also revealed the lineage-specificity of resistance profiles and resistance gene profiles. Overall, this study provides new insights and updated data support for understanding AMR and its genetic determinants in R. anatipestifer.

Abstract Duck Tembusu virus (DTMUV), an emerging pathogenic flavivirus, causes markedly decreased egg production in laying duck and neurological dysfunction and death in ducklings. Vaccination is currently the most effective means for prevention and control of DTMUV. In previous study, we have found that methyltransferase (MTase) defective DTMUV is attenuated and induces a higher innate immunity. However, it is not clear whether MTase-deficient DTMUV can be used as a live attenuated vaccine (LAV). In this study, we investigated the immunogenicity and immunoprotection of N7-MTase defective recombinant DTMUV K61A, K182A and E218A in ducklings. These three mutants were highly attenuated in both virulence and proliferation in ducklings but still immunogenic. Furthermore, a single-dose immunization with K61A, K182A or E218A could induce robust T cell responses and humoral immune responses, which could protect ducks from the challenge of a lethal-dose of DTMUV-CQW1. Together, this study provides an ideal strategy to design LAVs for DTMUV by targeting N7-MTase without changing the antigen composition. This attenuated strategy targeting N7-MTase may apply to other flaviviruses.

ABSTRACT Riemerella anatipestifer (R. anatipestifer) is a Gram-negative pathogen that causes significant economic losses to the farming industry. The polysaccharides that determine serotype are well understood in other bacteria; they have not yet been fully characterized in R. anatipestifer. In this study, we unexpectedly discovered a strain, RCAD0392, that is capable of cross-agglutination. Whole genome sequencing revealed base mutations disrupting a gene on its capsular polysaccharide synthesis gene cluster. By constructing homologous gene deletion mutants in CH-2, we demonstrated that deletion of this gene leads to altered serological phenotypes. India ink and transmission electron microscopy experiments revealed the disappearance of capsule on the surface of the bacteria, indicating the association of the gene with capsule synthesis. In addition, agar-gel precipitin tests showed that lipopolysaccharide binds to antisera of multiple serotypes, while the capsular polysaccharide only binds to the corresponding antisera. This suggests that capsular polysaccharide is the specific antigen that determines the serotype of R. anatipestifer. IMPORTANCE Riemerella anatipestifer (R. anatipestifer) is one of the most important veterinary pathogens with at least 21 serotypes. However, the exact polysaccharide(s) that determine R. anatipestifer serotype is still unknown. This study has provided a preliminary exploration of the relationship between capsular polysaccharides and serotyping in R. anatipestifer and suggests possible directions for further investigation of the genetic basis of serotypes in this bacterium.

ABSTRACT: Tigecycline is considered one of the “last resort antibiotics” for treating complex infections caused by multidrug-resistant (MDR) bacteria, especially for combating clinical resistant strains that produce carbapenemases. However, the tet(X4) gene, which carried by different plasmids can mediate high levels of bacterial resistance to tigecycline, was first reported in 2019. Here, we report the emergence of the plasmid-mediated tet(X4) in avian environment of Sichuan Province. A total of 21 tet(X4)-positive Escherichia coli (E. coli) strains were isolated and identified from avian samples in selected regions, with an isolation rate of 1.6% (21/1,286), and all of them were MDR strains. Multilocus Sequence Typing (MLST) method was used to classify the 21 tet(X4)-positive E. coli into the ST206, ST761, ST155, ST1638, ST542, and ST767 types, which also belong to the 3 phylogenetic subgroups A, B1, and C. Tet(X4) is located on mobile plasmids that can be efficiently and stably propagated. The results of fitness cost experiments showed that tet(X4)-positive plasmids may incur some fitness cost to host bacteria, but different tet(X4)-positive plasmids bring about differential fitness costs. Whole-genome sequencing further confirmed the tet(X4) gene can be located on IncX1-type plasmids and the core genetic structures are ISVsa3-rdmc-tet(X4) or rdmc-tet(X4)-ISVsa3, the former is a 7 copies tandem repeat structure. In this study, we isolated and identified tet(X4)-positive E. coli from the avian origin in Sichuan, analyzed the mobility of the tet(X4) by conjugational transfer and S1-PFGE, and evaluated the biological characteristics of the tet(X4)-positive plasmid using the results of conjugational frequency, plasmid stability, and fitness costs. Finally, combined with the third-generation whole-genome sequencing analysis, the molecular transmission characteristics of the tet(X4) were preliminarily clarified, providing a scientific basis for guiding veterinary clinical use in this area, as well as risk assessment and prevention of the transfer and spread of tigecycline resistant strains or genes.

ABSTRACT: Identification and analysis of the avian reovirus from black swan.Isolation of the strain through the chorioallantoic membrane route of duck embryos, identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. The complete genome of the ARV strain was obtained using next-generation sequencing technology.The isolated strain of ARV was named CD200801 and was identified through transmission electron microscopy and RT-PCR based on the ARV S2 gene. Experimental infection with CD200801 resulted in the death of ducklings with serious spleen and liver focal necrosis. BLAST analysis of CD200801 sequences showed a 35.5 to 98.6% similarity to a novel duck reovirus that was isolated in recent years. Phylogenetic analysis revealed that CD200801 was closely related to ARV isolates YL, GX-Y7, and XT-18.We report the first avian reovirus infection in the black swan. This study provides important new insights into the evolutionary relationships among different ARV strains and highlights the need for continued surveillance and monitoring of these viruses in both domestic and wild bird flocks. These findings have significant implications for the development of effective strategies for disease prevention and control in the poultry industry.

ABSTRACT: With the increasing number of ducks being raised and consumed, it is crucial to monitor the presence of multidrug resistant (MDR) bacteria in duck farming. Waterfowl, such as ducks, can contribute to the rapid dissemination of antibiotic resistance genes (ARGs). The objective of this study was to investigate the antimicrobial resistance (AMR), ARGs, and mobile genetic elements (MGEs), such as IS26, tbrC, ISEcp1 in Escherichia coli (E. coli) isolated from the intestinal contents of diseased ducks between 2021 and 2022 in Sichuan, Chongqing and Anhui, China. The AMR phenotypes of 201 isolated E. coli strains were determined using the minimum inhibitory concentrations (MICs) method. Subsequently, polymerase chain reaction and sequencing techniques were employed to screen for integron-integrase genes (intI1, intI2, intI3 genes), gene cassettes (GCs), MGEs, and ARGs. The results demonstrated that 96.5% of the E. coli isolates were resistant to at least 1 antibiotic, with 88.1% of the strains displaying MDR phenotype. The highest AMR phenotype observed was for trimethoprim-sulfamethoxazole (88.1%). Furthermore, class 1 and class 2 integrons were detected in 68.2% and 3.0% of all the isolates, respectively, whereas no class 3 integrons were found. Ten types of GCs were identified in the variable regions of class 1 and class 2 integrons. Moreover, 10 MGEs were observed in 46 combinations, with IS26 exhibiting the highest detection rate (89.6%). Among the 22 types of ARGs, tetA (77.1%) was the most frequently detected. In the conjugational transfer experiment, transconjugants were found to carry specific ARGs and MGEs, with their MIC values were significantly higher than those of recipient E. coli J53, indicating their status as MDR bacteria. This study emphasizes the necessity of monitoring MGEs, ARGs, and integrons in duck farms. It provides valuable insights into the complex formation mechanisms of AMR and may aid in preventing and controlling the spread of MDR bacteria in waterfowl breeding farm.

ABSTRACT: We investigated the cephalosporin resistance of Escherichia coli from waterfowl among different breeding mode farms. In 2021, we isolated 200 strains of E. coli from waterfowl feces samples collected from Sichuan, Heilongjiang, and Anhui provinces. The key findings are: Out of the 200 strains, 80, 80, and 40 strains were isolated from waterfowl feces samples in intensive, courtyard, and outdoor breeding mode farms, respectively. The overall positive rate of the ESBL phenotype, detecting by the double disk diffusion method, was 68.00% (136/200). In particular, the rates for intensive, courtyard, and outdoor breeding modes were 98.75%, 36.25%, and 70.00%, respectively. Results of MIC test showed drug resistance rates in the intensive breeding mode: 100.00% for cephalothin, 38.75% for cefoxitin, 100.00% for cefotaxime, and 100.00% for cefepime. In courtyard breeding mode, the corresponding rates were 100.00%, 40.00%, 63.75%, and 45.00%, respectively. In outdoor breeding mode, the corresponding rates were 100.00%, 52.50%, 82.50%, and 77.50%, respectively. The PCR results for blaCTX-M, blaTEM, blaOXA, and blaSHV showed the detection rate of blaCTX-M was highest at 75.50%, with blaCTX-M-55 is the main subtype gene, followed by blaTEM at 73.50%. We screened 58 donor strains carrying blaCTX-M-55, including 52 strains from the intensive breeding mode. These donor bacteria can transfer different plasmids to recipient E. coli J53, resulting in recipient bacteria acquiring cephalosporin resistance, and the conjugational transfer frequency ranged from 1.01 × 10-5 to 6.56 × 10-2. The transferred plasmids remained stable in recipient bacteria for up to several days without significant adaptation costs observed. During molecular typing of E. coli with conjugational transfer ability, the blaCTX-M-55 was found to be widely present in different ST strains with several phylogenetic groups. In summary, cephalosporin resistance of E. coli carried by waterfowl birds in intensive breeding mode farm was significantly higher than in courtyard and outdoor mode farms. The blaCTX-M-55 subtype gene was the prevalent ARGs and can be horizontally transferred through plasmids, which plays a key role in the spread of cephalosporin drug resistance.

Abstract Tembusu virus (TMUV) is an emerging flavivirus that has broken out in different regions of China. TMUV infection has been reported to induce autophagy in duck embryo fibroblast cells. However, the molecular mechanisms underlying this autophagy induction remain unclear. Here, we explored the interactions between autophagy and TMUV and the effects of the structural and nonstructural proteins of TMUV on autophagy in vitro. Among our results, TMUV infection enhanced autophagy to facilitate viral replication in HEK293T cells. After pharmacologically inducing autophagy with rapamycin (Rapa), the replication of TMUV increased by a maximum of 14-fold compared with the control group. To determine which TMUV protein primarily induced autophagy, cells were transfected with two structural proteins and seven nonstructural proteins of TMUV. Western blotting showed that nonstructural proteins 2B (NS2B) and 4 A (NS4A) of TMUV significantly induced the conversion of microtubule-associated protein 1 light chain 3 (LC3) from LC3-I to LC3-II in HEK293T cells. In addition, through immunofluorescence assays, we found that NS2B and NS4A significantly increased the punctate fluorescence of GFP-LC3-II. Furthermore, we found that both NS2B and NS4A interacted with polyubiquitin-binding protein sequestosome 1 (SQSTM1/p62) in a coimmunoprecipitation assay. Moreover, the autophagic degradation of p62 and LC3 mediated by NS2B or NS4A was inhibited by treatment with the autophagic flux inhibitor chloroquine (CQ). These results confirmed the vital effects of NS2B and NS4A in TMUV-induced complete autophagy and clarified the importance of complete autophagy for viral replication, providing novel insight into the relationship between TMUV and autophagy.

Abstract Duck hepatitis A virus type 1 (DHAV-1) is an acute, highly lethal infectious agent that infects ducklings and causes up to 95% mortality in ducklings up to 1 week of age, posing a significant economic threat to the duck farming industry. Previous studies have found that the proteolytic enzyme 3 C encoded by DHAV-1 can inhibit the IRF7 protein from blocking the upstream signaling pathway of the type I interferon to promote viral replication. However, there are still few studies on the mechanism of DHAV-1 in immune evasion. Here, we demonstrate that the DHAV-1 3CD protein can interact with IRF7 protein and reduce IRF7 protein expression without directly affecting IRF7 protein nuclear translocation. Further studies showed that the 3CD protein could reduce the expression of RIG-I protein without affecting its transcription level. Furthermore, we found that the 3CD protein interacted with the N-terminal structural domain of RIG-I protein, interfered with the interaction between RIG-I and MAVS, and degraded RIG-I protein through the proteasomal degradation pathway, thereby inhibiting its mediated antiviral innate immunity to promote DHAV-1 replication. These data suggest a novel immune evasion mechanism of DHAV-1 mediated by the 3CD protein, and the results of this experiment are expected to improve the understanding of the biological functions of the viral precursor protein and provide scientific data to elucidate the mechanism of DHAV-1 infection and pathogenesis.

ABSTRACT: Duck Tembusu virus (DTMUV), belonging to the Flaviviridae family, is a major virus that affects duck health in China. MicroRNAs (miRNAs) play an important role in viral replication. However, little is known about the function of miRNAs during DTMUV infection. Here, the host miR-146b-5p was found to regulate DTMUV replication. When DTMUV infected duck embryo fibroblasts (DEFs), the expression levels of miR-146b-5p increased significantly over time. Moreover, the viral RNA copies, E protein expression levels and virus titers were all upregulated when miR-146b-5p was overexpressed in DEFs. The opposite results were also observed upon knockdown of miR-146b-5p in DEFs. To explore the mechanism by which miR-146b-5p promoted DTMUV replication, mass spectrometry, and RNA pull-down assays were employed. Ribosomal protein S14 (RPS14), a component of 40S ribosomal proteins, was identified to interact with miR-146b-5p. In addition, the relative mRNA expression levels of RPS14 gene were negatively modulated by miR-146b-5p. Subsequently, it was found that overexpression of RPS14 could decrease the replication of DTMUV, and the reverse results were also detected by knockdown of RPS14. In conclusion, this study revealed that miR-146b-5p promoted DTMUV replication by targeting RPS14, which provides a new mechanism by which DTMUV evades host defenses and a new direction for further antiviral strategies development.

ABSTRACT The bacterium Riemerella anatipestifer requires iron for growth, but the mechanism of iron uptake is not fully understood. In this study, we disrupted the Feo system and characterized its function in iron import in R. anatipestifer ATCC 11845. Compared to the parent strain, the growth of the ΔfeoA, ΔfeoB, and ΔfeoAB strains was affected under Fe3+-limited conditions, since the absence of the feo system led to less intracellular iron than in the parent strain. In parallel, the ΔfeoAB strain was shown to be less sensitive to streptonigrin, an antibiotic that requires free iron to function. The sensitivity of the ΔfeoAB strain to hydrogen peroxide was also observed to be diminished compared with that of the parent strain, which could be related to the reduced intracellular iron content in the ΔfeoAB strain. Further research revealed that feoA and feoB were directly regulated by iron through the Fur regulator and that the transcript levels of feoA and feoB were significantly increased in medium supplemented with 1 mM MnCl2, 400 μM ZnSO4, and 200 μM CuCl2. Finally, it was shown that the ΔfeoAB strain of R. anatipestifer ATCC 11845 was significantly impaired in its ability to colonize the blood, liver, and brain of ducklings. Taken together, these results demonstrated that FeoAB supports ferrous iron acquisition in R. anatipestifer and plays an important role in R. anatipestifer colonization. IMPORTANCE In Gram-negative bacteria, the Feo system is an important ferrous iron transport system. R. anatipestifer encodes an Feo system, but its function unknown. As iron uptake may be required for oxidative stress protection and virulence, understanding the contribution of iron transporters to these processes is crucial. This study showed that the ΔfeoAB strain is debilitated in its ability to import iron and that its intracellular iron content was constitutively low, which enhanced the resistance of the deficient strain to H2O2. We were surprised to find that, in addition to responding to iron, the Feo system may play an important role in sensing manganese, zinc, and copper stress. The reduced colonization ability of the ΔfeoAB strain also sheds light on the role of iron transporters in host-pathogen interactions. This study is important for understanding the cross talk between iron and other metal transport pathways, as well as the pathogenic mechanism in R. anatipestifer.

ABSTRACT Duck plague virus (DPV) is a member of Alphaherpesvirus genus and poses a major threat to waterfowl breeding. Genetic engineered vaccines that are capable of distinguishing naturally infected from vaccine-immunized animals are useful for eradicating duck plague. In this study, reverse genetics was used to develop an ICP27-deficient strain (CHv-ΔICP27), and its potential as a marker vaccination candidate was evaluated. The results showed that the CHv-ΔICP27 generated in this study exhibited good genetic stability in vitro and was highly attenuated both in vivo and in vitro. The level of neutralizing antibody generated by CHv-ΔICP27 was comparable to that induced by a commercial DPV vaccine, suggesting that it could protect ducks from virulent DPV attack. By using molecular identification techniques such as PCR, restriction fragment length polymorphism, immunofluorescence, Western blotting, and others, it is possible to differentiate the CHv-ΔICP27 from wild-type strains. Moreover, ICP27 can also be a potential target for the genetic engineering vaccine development of alphavirus or perhaps the entire herpesvirus family members due to the highly conservative of ICP27 protein in all herpesvirus family members. IMPORTANCE The development of distinguishable marker vaccines from natural infection is a key step toward eradicating duck plague. Here, we generated a recombinant DPV that carries an ICP27 deletion marker that could be easily distinguished from wild-type strain by molecular biological methods. It was highly attenuated in vitro and in vivo and could provide comparable protection to ducks after a single dose of immunizations, as commercial vaccines did. Our findings support the use of the ICP27-deficient virus as a marker vaccine for DPV control and future eradication.

Abstract An alphaherpesvirus carries dozens of viral proteins in the envelope, tegument and capsid structure, and each protein plays an indispensable role in virus adsorption, invasion, uncoating and release. After infecting the host, a virus eliminates unfavourable factors via multiple mechanisms to escape or suppress the attack of the host immune system. Post-translational modification of proteins, especially phosphorylation, regulates changes in protein conformation and biological activity through a series of complex mechanisms. Many viruses have evolved mechanisms to leverage host phosphorylation systems to regulate viral protein activity and establish a suitable cellular environment for efficient viral replication and virulence. In this paper, viral protein kinases and the regulation of viral protein function mediated via the phosphorylation of alphaherpesvirus proteins are described. In addition, this paper provides new ideas for further research into the role played by the post-translational modification of viral proteins in the virus life cycle, which will be helpful for understanding the mechanisms of viral infection of a host and may lead to new directions of antiviral treatment.

Abstract Duck hepatitis A virus type 1 (DHAV-1) is one of the main pathogens responsible for death in ducklings. Autophagy is a catabolic process that maintains cellular homeostasis, and the PI3KC3 protein plays an important role in the initiation of autophagy. DHAV-1 infection induces autophagy in duck embryo fibroblasts (DEFs) but the molecular mechanism between it and autophagy has not been reported. First, we determined that DHAV-1 infection induces autophagy in DEFs and that autophagy induction is dependent on the integrity of viral proteins by infecting DEFs with UV-inactivated or heat-inactivated DHAV-1. Then, in experiments using the pharmacological autophagy inducer rapamycin and the autophagy inhibitor chloroquine, autophagy inhibition was shown to reduce intracellular and extracellular DHAV-1 genome copies and viral titres. These results suggest that autophagy activated by DHAV-1 infection in DEFs affects DHAV-1 proliferation and extracellular release. Next, we screened the autophagy-inducing effects of the DHAV-1 structural proteins VP0, VP3, and VP1 and found that all DHAV-1 structural proteins could induce autophagy in DEFs but not the full autophagic flux. Finally, we found that VP1 promotes protein expression of PI3KC3 and Beclin1 by western blot experiments and that VP1 interacts with PI3KC3 by co-immunoprecipitation experiments; moreover, 3-MA-induced knockdown of PI3KC3 inhibited VP1 protein-induced autophagy in DEFs. In conclusion, the DHAV-1 structural protein VP1 regulates the PI3KC3 complex by interacting with PI3KC3 to induce autophagy in DEFs.

Abstract Duck Tembusu virus (DTMUV) is a pathogenic flavivirus that has caused enormous economic losses in Southeast Asia. Our previous study showed that DTMUV could induce duck embryo fibroblast (DEF) apoptosis, but the specific mechanism was not clear. In this study, we confirmed that DTMUV could induce the apoptosis of DEFs by DAPI staining and TUNEL staining. Furthermore, we found that the expression levels of cleaved-caspase-3/7/8/9 were significantly upregulated after DTMUV infection. After treatment of cells with an inhibitor of caspase-8 or caspase-9, DTMUV-induced apoptosis rates were significantly decreased, indicating that the caspase-8-mediated death receptor apoptotic pathway and caspase-9-mediated mitochondrial apoptotic pathway were involved in DTMUV-induced apoptosis. Moreover, we found that DTMUV infection not only caused the release of mitochondrial cytochrome C (Cyt C) and the downregulation of the apoptosis-inhibiting protein Bcl-2 but also reduced the mitochondrial membrane potential (MMP) and the accumulation of intracellular reactive oxygen species (ROS). Key genes in the mitochondrial apoptotic pathway and death receptor apoptotic pathway were upregulated to varying degrees, indicating the activation of the mitochondrial apoptosis pathway and death receptor apoptosis pathway. In conclusion, this study clarifies the molecular mechanism of DTMUV-induced apoptosis and provides a theoretical basis for revealing the pathogenic mechanism of DTMUV infection.

Abstract Background Duck plague virus (DPV), belonging to herpesviruses, is a linear double-stranded DNA virus. There are many reports about the outbreak of the duck plague in a variety of countries, which caused huge economic losses. Recently, increasing reports revealed that multiple long non-coding RNAs (lncRNAs) can possess great potential in the regulation of host antiviral immune response. Furthermore, it remains to be determined which specific molecular mechanisms are responsible for the DPV-host interaction in host immunity. Here, lncRNAs and mRNAs in DPV infected duck embryonic fibroblast (DEF) cells were identified by high-throughput RNA-sequencing (RNA-seq). And we predicted target genes of differentially expressed genes (DEGs) and formed a complex regulatory network depending on in-silico analysis and prediction. Result RNA-seq analysis results showed that 2921 lncRNAs were found at 30 h post-infection (hpi). In our study, 218 DE lncRNAs and 2840 DE mRNAs were obtained in DEF after DPV infection. Among these DEGs and target genes, some have been authenticated as immune-related molecules, such as a Macrophage mannose receptor (MR), Anas platyrhynchos toll-like receptor 2 (TLR2), leukocyte differentiation antigen, interleukin family, and their related regulatory factors. Furthermore, according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, we found that the target genes may have important effects on biological development, biosynthesis, signal transduction, cell biological regulation, and cell process. Also, we obtained, the potential targeting relationship existing in DEF cells between host lncRNAs and DPV-encoded miRNAs by software. Conclusions This study revealed not only expression changes, but also the possible biological regulatory relationship of lncRNAs and mRNAs in DPV infected DEF cells. Together, these data and analyses provide additional insight into the role of lncRNAs and mRNAs in the host's immune response to DPV infection.

ABSTRACT: Duck plague is an acute, febrile, and septic infectious disease caused by duck plague virus (DPV), which causes serious harm to the duck industry in China. Ducks latently infected with DPV display a clinically healthy state, which is one of the epidemiological characteristics of duck plague. In the present study, to rapidly distinguish vaccine-immunized ducks from wild virus-infected ducks during production, a PCR assay based on the newly identified LORF5 fragment was developed to effectively and accurately identify viral DNA in cotton swab samples and was used to assess artificial infection models and clinical samples. The results showed that the established PCR method had good specificity and that only the virulent and attenuated DNA of duck plague virus was specifically amplified, as the results for the detection of common duck pathogens (duck hepatitis B virus, duck Tembusu virus, duck hepatitis A virus type 1, novel duck reovirus, Riemerella anatipestifer, Pasteurella multocida, and Salmonella) were negative. The amplified fragments of virulent and attenuated strains were 2,454 bp and 525 bp, and their minimum detection amounts were 0.46 pg and 46 pg, respectively. The detection rate of the virulent and attenuated DPV strains in duck oral and cloacal swabs was lower than that of the gold standard PCR method (GB-PCR, which is unable to distinguish virulent and attenuated strains), and cloacal swabs from clinically healthy ducks were more suitable for detection than oral swabs. In conclusion, the PCR assay established in the present study can be used as a simple and effective method for the clinical screening of ducks that are latently infected with virulent strains of DPV and shedding virus, which can provide technical support for the elimination of duck plague from duck farms.

ABSTRACT: Duck plague virus (DPV) is a typical DNA virus of waterfowl, it causes huge economic losses to the duck industry due to the higher mortality and lower egg production. The disease is one of the frequent epidemics and outbreaks on duck farms because present vaccines could not provide complete immunity and there are no specific antiviral drugs available. Therefore, the development of antiviral drugs is urgently needed. In this study, we evaluated the antiviral activity of BX795, a specific kinase inhibitor of 3-phosphoinositide-dependent kinase 1 (PDK1), protein kinase B (AKT) and Tank binding kinase 1 (TBK1), against DPV in different duck cells. Our study demonstrated that BX795 reveals prominent antiviral activity in a dose-dependent manner in different types of duck cells. Time-addition and antiviral duration analysis uncovered that BX795 inhibits viral infection therapeutically and its antiviral activity lasts longer than 96 h. Further studies have shown that BX795 prevents cell-to-cell spread of the DPV rather than affects other stage of viral life cycle. Mechanistically, BX795 can inhibit DPV US3 kinase activity, reduce the phosphorylation of US3 substrates, and prevent the interaction between US3 and UL47. Taking together, our study demonstrated BX795, which disrupts the viral kinase activity, is a candidate antiviral agent for DPV.

ABSTRACT: Duck circovirus genotype 2 (DuCV2) belongs to the genus Circovirus, family Circoviridae. It can generally cause lymphocyte atrophy and necrosis in ducks, which leads to immunosuppression. The function of the DuCV2 open reading frame 3 (ORF3) protein in viral pathogenesis in host cells remains unclear. Therefore, a series of studies based on ORF3 of the isolate DuCV GH01 strain (belonging to DuCV2) were carried out in duck embryo fibroblasts (DEFs) in this study. The results showed that the ORF3 protein could induce nuclear shrinkage and fragmentation in DEFs. Chromosomal DNA breakage was observed by TUNEL assay. The expression levels of caspase-related genes showed that ORF3 primarily promoted caspase 3 and caspase 9 expression. Furthermore, the protein expression levels of cleaved caspase 3 and cleaved caspase 9 in DEFs were enhanced by ORF3. Thus, ORF3 may activate the mitochondrial apoptosis pathway. When the 20 amino acid residues at the C-terminus of ORF3 (ORF3ΔC20) were deleted, the apoptosis rates were decreased. Moreover, compared to ORF3, ORF3ΔC20 downregulated the mRNA levels of cytochrome c (Cyt c), poly ADP-ribose polymerase (PARP) and apoptosis protease activating factor 1 (Apaf-1), which are the key molecules in the mitochondrial apoptotic pathway. Further study showed that ORF3ΔC20 could reduce the mitochondrial membrane potential (MMP). This study suggested that the DuCV2 ORF3 protein may primarily activate apoptosis through the mitochondrial pathway in DEFs, and this function is ORF3 C20 dependent.

Abstract Background Duck hepatitis A virus type 1 (DHAV-1) is one of the most serious pathogens endangering the duck industry. However, there are few studies on the regulation of the cell cycle by DHAV-1. Methods In this study, flow cytometry was applied to analyze the effect of DHAV-1 infection on the cell cycle of duck embryo fibroblasts (DEFs). Subsequently, we analyzed the effects of cell cycle phases on DHAV-1 replication by real-time reverse transcriptase quantitative PCR (real-time RT-qPCR). Results Flow cytometry data analysis found that DEFs in the S phase increased by 25.85% and 54.21% at 24 h and 48 h after DHAV-1 infection, respectively. The levels of viral RNA detected by real-time RT-qPCR were higher in the DEFs with synchronization in the S phase or G0/G1 phase than in the control group. However, there was no difference in viral copy number between the G2/M phase arrest and control groups. In addition, non-structural protein 3D of DHAV-1 significantly increased cells in the S phase, indicating that 3D protein is one of the reasons for the cell cycle arrest in the S phase. Conclusions In summary, DHAV-1 infection induces the cell cycle arrest of DEFs in the S phase. Both S phase and G0/G1 phase synchronization facilitate the replication of DHAV-1, and 3D protein is one of the reasons for the S phase arrest.

Abstract Live-attenuated vaccines (LAVs) represent a promising approach for flavivirus vaccine development. In the present study, we demonstrated a method for generating flavivirus LAVs based on breaking spatially and temporally regulated C-prM cleavage to disturb the viral assembly process, using an avian flavivirus (Tembusu virus) as the model. Using reverse genetics technology, we successfully generated two recombinant viruses (CQW1-IRES-mC and CQW1-MINI-mC) with bicistronic genomic RNA in which native capsid genes were deleted and instead expressed in the 3’UTR under the control of an internal ribosome entry site (IRES) or minimum IRES. Both viruses showed a significantly attenuated phenotype in vitro due to impaired viral assembly, and the engineered mutations were genetically stable in vitro within ten passages. Importantly, their virulence was also highly attenuated in ducklings and suckling mice and did not cause any overt clinical symptoms or mortality. In addition, a single dose of immunization with any of these mutant viruses could completely protect ducklings from a lethal challenge, and no viremia was detected after immunization and challenge, even though the viruses induced a relatively moderate immune response in terms of the T-lymphocytes proliferative response and the level of neutralization antibodies compared with that obtained with the wild-type virus. Besides, a recombinant virus ectopically expressing the prM-E protein was also generated in the present study, but this virus was too attenuated with severely decreased proliferation. Our results indicated that the use of a recombinant flavivirus that ectopically expresses structural proteins could be an effective and universal method for flavivirus LAVs development.

Abstract Duck Tembusu virus (DTMUV), a member of the family Flaviviridae and an economically important pathogen with a broad host range, leads to markedly decreased egg production. However, the molecular mechanism underlying the host-DTMUV interaction remains unclear. Here, we performed high-throughput RNA sequencing (RNA-Seq) to study the dynamic changes in host gene expression at 12, 24, 36, 48 and 60 h post-infection (hpi) in duck embryo fibroblasts (DEF) infected with DTMUV. A total of 3129 differentially expressed genes (DEG) were identified after DTMUV infection. Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these DEG were associated with multiple biological functions, including signal transduction, host immunity, virus infection, cell apoptosis, cell proliferation, and pathogenicity-related and metabolic process signaling pathways. This study analyzed viral infection and host immunity induced by DTMUV infection from a novel perspective, and the results provide valuable information regarding the mechanisms underlying host-DTMUV interactions, which will prove useful for the future development of antiviral drugs or vaccines for poultry, thus benefiting the entire poultry industry.

Epigenetics modulates expression levels of various important genes in both prokaryotes and eukaryotes. These epigenetic traits are heritable without any change in genetic DNA sequences. DNA methylation is a universal mechanism of epigenetic regulation in all kingdoms of life. In bacteria, DNA methylation is the main form of epigenetic regulation and plays important roles in affecting clinically relevant phenotypes, such as virulence, host colonization, sporulation, biofilm formation et al. In this review, we survey bacterial epigenomic studies and focus on the recent developments in the structure, function, and mechanism of several highly conserved bacterial DNA methylases. These methyltransferases are relatively common in bacteria and participate in the regulation of gene expression and chromosomal DNA replication and repair control. Recent advances in sequencing techniques capable of detecting methylation signals have enabled the characterization of genome-wide epigenetic regulation. With their involvement in critical cellular processes, these highly conserved DNA methyltransferases may emerge as promising targets for developing novel epigenetic inhibitors for biomedical applications.

ABSTRACT: Duck plague virus (DPV) is a pathogen causing duck plague and has caused huge economic losses in poultry industry. In our previous report, US3 gene deletion from DPV genome seriously impaired virus replication. In this study, we constructed a US3 kinase-inactive mutant (US3K213A) to further explore the function of US3 protein (pUS3) in DPV. Our results showed that the loss of pUS3 kinase activity caused lower viral titers, smaller plaque sizes and a blockage of capsids nuclear egress including primary enveloped virion (PEV) accumulation compared to the parental virus infection. It indicates that the effects of DPV pUS3 on viral propagation depended on its kinase activity. In addition, we conducted electron microscopy analysis to show the outer nuclear membrane (ONM) evaginations and the nuclear envelope (NE) deep invagination in US3K213A-infected cells. Finally, an irregular distribution of pUL31/pUL34 in the NE in △US3- and US3K213A-infected cells and an interaction of pUS3 and pUL31 were found, which suggests that pUS3 potentially targets pUL31 and regulates the localization of pUL31/pUL34 to promote nucleocapsids egress through its kinase activity.

ABSTRACT: Riemerella anatipestifer (R. anatipestifer, RA) is an infectious pathogen that causes septicemia and polyserositis in ducks. Our previous studies showed that RA CH-1 ∆fur was significantly attenuated in ducklings, which highlights the potential of this strain as a live attenuated vaccine. In this study, it was shown that infection with 109 CFU of the fur mutant did not cause any clinical symptoms or significant histological lesions in 3-day-old ducklings and that the bacteria were readily cleared by the host within 3 d. Compared with the nonvaccinated group, the group inoculated with the mutant strain RA CH-1 ∆fur exhibited protection of ducklings against a high-dose (2.28 × 1010 CFU) challenge with the wild-type strain RA CH-1. Moreover, the average body weights and body weight gains of the Δfur-inoculated group were not significantly affected by the challenge. Further analysis revealed that RA CH-1 ∆fur elicited higher IgY titers and that the serum antibody levels persisted for at least 49 d after immunization. Overall, our study showed that RA CH-1 ∆fur is a safe and effective vaccine candidate that is expected to play an important role in RA CH-1 infection prevention in the duck industry.

ABSTRACT Recently, a new strategy for attenuating RNA viruses by redirecting their evolution in sequence space was confirmed for Enterovirus and Influenza viruses. Using avian flavivirus as a model, the 69 serine and 53 leucine codons on the E-NS1 genes were modified to change evolutionary direction of the viral sequence space. This means that all codons encoding serine or leucine residues were substituted with codons that are only one base different from the three stop codons, resulting in the initial position of the virus genome in sequence space being closer to the detrimental areas to achieve attenuation by reducing viral adaptability. The growth curve and plaque size of CQW1-one-to-stop (CQW1-OTS) were similar to those of CQW1-wild type (CQW1-WT) in vitro, but attenuated proliferation was detected when treated with a mutagenic reagent (ribavirin). However, comparably high CQW1-OTS and CQW1-WT lethality rates were detected in 9-day-old duck embryos and 5-day-old ducklings, suggesting that this strategy works but with limitations. With that in mind, homologous hosts in nonsensitive age (25-day-old ducks) and heterologous hosts (3-week-old Kunming mice) were employed to investigate if CQW1-OTS was attenuated under host selection pressure. Minimal attenuation of CQW1-OTS in elder ducks and apparent attenuation in mice were reported, providing reduced viral titers, mild clinical signs, and lower specific infectivity. Collectively, we experimentally demonstrate that the attenuation strategy of redirecting virus evolution in sequence space works for flavivirus. Redirection of the virus is attenuated only under some outside pressure, such as heterologous hosts or antiviral drugs treatment, limiting its usage in flaviviruses. IMPORTANCE Flaviviruses are medically important arboviruses that threaten public health, but no approved treatments are currently available. Vaccines prevent flavivirus infection. We employed duck Tembusu virus (TMUV), a mosquito-borne flavivirus, to evaluate virus redirection. TMUV is native to birds and could infect mice by intracerebral injection, making it an experimental animal model to study flavivirus characteristics in vivo. The 69 serine and 53 leucine codons on the E-NS1 proteins of CQW1 were synonymously substituted to change evolutionary direction of the virus in sequence space. In vitro mutagen reagent treatment suppressed CQW1-OTS viral multiplication, but in vivo attenuation depended on host selective pressure. CQW1-OTS viral attenuation was observed in older ducks but not sensitive ducklings; considerable attenuation was also observed in heterogenous host (mice), which provides more selective pressure on viruses. Collectively, these data indicated that there are very important preconditions for application of evaluating whether this strategy shows application prospects in novel flavivirus vaccine development.

ABSTRACT: Duck plague virus (DPV) pUL48 is a homologous of herpes simplex virus VP16, and some studies have shown that VP16 is essential for viral replication and proliferation, but there are few studies on DPV pUL48. Therefore, in order to study the function of pUL48 protein, we constructed a UL48-deleted mutant (DPV-BAC-∆UL48) that completely reemoved the UL48 gene from the DPV BAC genome and the revertant virus (DPV-BAC-∆UL48R) by using the 2-step red recombination system. Compared with the parental virus (DPV-BAC) and the revertant virus, the titer of UL48-deleted mutant was reduced by more than 38.2%, and the efficiency of producing infectious virions was significantly reduced. In addition, the average size of plaques produced by UL48-deleted mutant was about 30% smaller than that of the parental and revertant viruses, suggesting that pUL48 protein affected the cell-to-cell transmission of DPV. Finally, pharmacological inhibition assay showed that pUL48 is a late protein of DPV. In this study, we found that UL48, as a late gene, plays an important role in viral replication by affecting the formation of DPV infectious virion, virus cell-to-cell transmission, and viral genome transcription, which may provide some help for the study of the function of DPV pUL48 protein and the prevention and control of DPV.

Goose astrovirus (GAstV, genus Avian Astrovirus, family Astrovirus) was first discovered in 2005, but was not considered as a pathogen of gosling gout until 2016. Since then, goose astrovirus has erupted in Chinese goslings, causing at most 50% of gosling deaths. By December 2022, the disease had become epidemic and prevailed in goose farms in Jiangsu, Shandong, Anhui, Henan, Guangdong, Liaoning, Sichuan and other places in China. The disease mainly affects goslings within 3 weeks old. The typical symptoms of goose astrovirus are large deposits of urate in the viscera, joint cavity and ureter surface of infected goslings. Goose astrovirus infection can trigger high levels of iNOS, limiting goose astrovirus replication. The ORF2 domain P2 of the goose astrovirus activates the OASL protein, limiting its replication. Goose astrovirus can also activate pattern recognition receptors (RIG-I, MDA-5, TLR-3), causing an increase in MHC-Ia, MHC-Ib and CD81 mRNA, activating humoral and cellular immunity, thereby hindering virus invasion. Goose astrovirus also regulates the activation of IFNs and other antiviral proteins (Mx1, IFITM3, and PKR) in the spleens and kidneys to inhibit viral replication. The innate immune response process in goslings also activates TGF-β, which may be closely related to the immune escape of goose astrovirus. Gaining insight into the infection and innate immune mechanism of goose astrovirus can help researchers study and prevent the severe disease in goslings better.

Abstract Pasteurella multocida infection frequently causes fowl cholera outbreaks, leading to huge economic losses to the poultry industry worldwide. This study developed a novel live attenuated P. multocida vaccine strain for ducks named PMZ2 with deletion of the gatA gene and first four bases of the hptE gene, both of which are required for the synthesis of the lipopolysaccharide (LPS) outer core. PMZ2 produced a truncated LPS phenotype and was highly attenuated in ducks with a > 105-fold higher LD50 than the wild-type strain. PMZ2 colonized the blood and organs, including the spleen, liver and lung, at remarkably reduced levels, and its high dose of oral infection did not cause adverse effects on body temperatures and body weights in ducks. To evaluate the vaccine efficacy of the mutant, ducklings were inoculated orally or intranasally with PMZ2 or PBS twice and subsequently subjected to a lethal challenge. Compared with the PBS control, PMZ2 immunization stimulated significantly elevated serum IgG, bile IgA and tracheal IgA responses, especially after the boost immunization in both the oral and intranasal groups, and the induced serum had significant bactericidal effects against the wild-type strain. Furthermore, the two PMZ2 immunization groups exhibited alleviated tissue lesions and significantly decreased bacterial loads in the blood and organs compared with the PBS group post-challenge. All the ducks in the PMZ2 oral and intranasal groups survived the challenge, while 70% of ducks in the PBS group succumbed to the challenge. Thus, the P. multocida mutant with mutation of the gatA gene and part of the hptE gene proved to be an effective live attenuated vaccine candidate for prevention of fowl cholera in ducks.

Abstract Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic pattern recognition receptors that initiate innate antiviral immunity. Recent reports found that duck RLRs significantly restrict duck plague virus (DPV) infection. However, the molecular mechanism by which DPV evades immune responses is unknown. In this study, we first found that the DPV UL41 protein inhibited duck interferon-β (IFN-β) production mediated by RIG-I and melanoma differentiation-associated gene 5 (MDA5) by broadly downregulating the mRNA levels of important adaptor molecules, such as RIG-I, MDA5, mitochondrial antiviral signalling protein (MAVS), stimulator of interferon gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF) 7. The conserved sites of the UL41 protein, E229, D231, and D232, were responsible for this activity. Furthermore, the DPV CHv-BAC-ΔUL41 mutant virus induced more duck IFN-β and IFN-stimulated genes (Mx, OASL) production in duck embryo fibroblasts (DEFs) than DPV CHv-BAC parent virus. Our findings provide insights into the molecular mechanism underlying DPV immune evasion.

ABSTRACT Duck plague caused by duck plague virus (DPV) is one of the main diseases that seriously endangers the production of waterfowl. DPV possesses a large genome consisting of 78 open reading frames (ORFs), and understanding the function and mechanism of each encoded protein in viral replication and pathogenesis is the key to controlling duck plague outbreaks. US1 is one of the two genes located in the repeat regions of the DPV genome, but the function of its encoded protein in DPV replication and pathogenesis remains unclear. Previous studies found that the US1 gene or its homologs exist in almost all alphaherpesviruses, but the loci, functions, and pathogenesis of their encoded proteins vary among different viruses. Here, we aimed to define the roles of US1 genes in DPV infection and pathogenesis by generating a double US1 gene deletion mutant and its revertant without any mini-F cassette retention. In vitro and in vivo studies found that deletion of both copies of the US1 gene significantly impaired the replication, gene expression, and virulence of DPV, which could represent a potential candidate vaccine strain for the prevention of duck plague. IMPORTANCE Duck plague virus contains nearly 80 genes, but the functions and mechanisms of most of the genes have not yet been elucidated, including those of the newly identified immediate early gene US1. Here, we found that US1 deletion reduces viral gene expression, replication, and virus production both in vitro and in vivo. This insight defines a fundamental role of the US1 gene in DPV infection and indicates its involvement in DPV transcription. These results provide clues for the study of the pathogenesis of the US1 gene and the development of attenuated vaccines targeting this gene.

DNAzymes effectively inhibit the expression of viral genes. Duck hepatitis A virus type-1 (DHAV-1) genomic RNA carries an internal ribosome entry site (IRES). The IRES initiates the translation of DHAV-1 via a mechanism that differs from that of cap-dependent translation. Therefore, it is an attractive target for the treatment of DHAV-1. In this study, we designed 6 DNAzymes (Dzs) specifically targeting 300–618 nt sequence in the DHAV-1 5′untranslated region (UTR; a predicted IRES-like element). In the presence of divalent metal ions, three designed DNAzymes (DZ369, DZ454, and DZ514) efficiently cleaved the 300–618 nt sequence of the DHAV-1 5′UTR RNA. The activity of the Dzs was particularly dependent on Mg2+ ions. Subsequently, the translation inhibitory activity of these Dzs was determined by western blotting experiments. The Dzs effectively inhibited the translation mediated by the 300–618 nt of DHAV-1 5′UTR in duck embryo fibroblasts (DEFs). Importantly, DZ454 showed the strongest inhibitory effect, and its inhibition was time and dose dependent. However, none of the Dzs showed significant inhibition of cap-dependent translation. These results suggest that these Dzs show specificity for target RNA. Moreover, DZ454 inhibited the replication of DHAV-1. In conclusion, the designed DNAzymes can be used as inhibitors of DHAV-1 RNA translation and replication, providing new insights useful for the development of anti-DHAV-1 drugs.

ABSTRACT: Identification and analysis of the antimicrobial resistance of Pasteurella aerogenes (P. aerogenes) isolated from poultry. For susceptibility testing in accordance with the CLSI, plasmids were extracted via alkaline lysis and transferred by CaCl2 treatment. Genomic DNA of a representative P. aerogenes isolate was subjected to whole genome sequencing. CCCP was utilized to determine whether SF190908 contains an efflux pump. The blaVEB gene was ligated with the pET-28 plasmid and transferred to Escherichia coli to verify it as an ESBL gene. SF190908 isolated from poultry was identified as P. aerogenes based upon biochemical and 16s rRNA results. The isolate showed high MIC values for eight antimicrobials. Sequencing results showed that the mobile element-mediated antimicrobial resistance gene cluster conferred antimicrobial resistance on the strain, and a single 5,105-bp plasmid, designated pRCAD0752PA-1, was isolated. Four antimicrobial resistance gene clusters were identified in the SF190908 chromosome; one antimicrobial resistance gene cluster carried the blaVEB gene, which was verified as ESBL according to the CLSI and was detected in Pasteurellaceae for the first time, to the best of our knowledge. The efflux pump may confer antimicrobial resistance to SF190908. P. aerogenes isolated from poultry showed resistance genes encoded in mobile elements that confer multi-antimicrobial resistance to SF190908. The antimicrobial-resistant plasmid pRCAD0752PA-1 was isolated in SF190908 and conferred resistance to florfenicol. This study indicates an urgent need to increase efforts to monitor the spread of P. aerogenes multi-antimicrobial-resistant strains and plasmids, especially in newly discovered at-risk species such as poultry.

Upon infection, the herpes viruses create a cellular environment suitable for survival, but innate immunity plays a vital role in cellular resistance to viral infection. The UL13 protein of herpesviruses is conserved among all herpesviruses and is a serine/threonine protein kinase, which plays a vital role in escaping innate immunity and promoting viral replication. On the one hand, it can target various immune signaling pathways in vivo, such as the cGAS-STING pathway and the NF-κB pathway. On the other hand, it phosphorylates regulatory many cellular and viral proteins for promoting the lytic cycle. This paper reviews the research progress of the conserved herpesvirus protein kinase UL13 in immune escape and viral replication to provide a basis for elucidating the pathogenic mechanism of herpesviruses, as well as providing insights into the potential means of immune escape and viral replication of other herpesviruses that have not yet resolved the function of it.

ABSTRACT: Bacterial antimicrobial resistance (AMR) continues to develop, with the horizontal transfer of antibiotic resistance genes (ARGs) through plasmids playing a major role. Recently, the antimicrobial resistance of R. anatipestifer has become increasingly severe, jeopardizing the development of the poultry industry. In this study, we used PromethION to determine the whole genome sequence of R. anatipestifer RCAD0416, a multidrug-resistant isolate from China. We detected a plasmid in the isolate. We named the plasmid pRCAD0416RA-1; the plasmid was 37356 bp in size with 36 putative open reading frames and included the blaOXA-347, floR, tet(X), ermF, ereD, and AadS resistance genes. Most resistance genes might be obtained from R. anatipestifer HXb2. Mobile elements and floR might be transmitted by plasmid pB18–2 from Acinetobacter indicus, and the ICEPg6Chn1 mobile elements can be transmitted from Proteus genomosp. The plasmid pRCAD0416RA-1 was transferred to Escherichia coli K-12 × 7232 via electroporation. Subsequent antimicrobial sensitivity tests (AST) showed a noticeable levels of antimicrobial resistance to β-lactams (4–8 fold), tigecycline (8 fold), and florfenicol (8 fold). These types of antibiotics are in common clinical use. The purpose of this article is to elucidate the basic characteristics of pRCAD0416RA-1 and the level of resistance mediated by blaOXA-347, floR, and tet(X).

ABSTRACT Tembusu virus (TMUV), an avian mosquito-borne flavivirus, was first identified from Culex tritaeniorhynchus in 1955. To validate the effects of the 3′-untranslated region (3′UTR) in viral host-specific adaptation, we generated a set of chimeric viruses using CQW1 (duck strain) and MM 1775 (mosquito strain) as backbones with heterogeneous 3′UTRs. Compared with rMM 1775, rMM-CQ3′UTR (recombinant MM 1775 virus carrying the 3′UTR of CQW1) exhibited enhanced proliferation in vitro, with peak titers increasing by 5-fold in duck embryonic fibroblast (DEF) cells or 12-fold in baby hamster kidney (BHK-21) cells; however, the neurovirulence of rMM-CQ3′UTR was attenuated in 14-day-old Kunming mice via intracranial injection, with slower weight loss, lower mortality, and reduced viral loads. In contrast, rCQ-MM3′UTR showed similar growth kinetics in vitro and neurovirulence in mice compared with those of rCQW1. Then, the Stem-loop I (SLI) structure, which showed the highest variation within the 3′UTR between CQW1 and MM 1775, was further chosen for making chimeric viruses. The peak titers of rMM-CQ3′UTRSLI displayed a 15- or 4-fold increase in vitro, and the neurovirulence in mice was attenuated, compared with that of rMM 1775; rCQ-MM3′UTRSLI displayed comparable multiplication ability in vitro but was significantly attenuated in mice, in contrast with rCQW1. In conclusion, we demonstrated that the TMUV SLI structure of the 3′UTR was responsible for viral host-specific adaptation of the mosquito-derived strain in DEF and BHK-21 cells and regulated viral pathogenicity in 14-day-old mice, providing a new understanding of the functions of TMUV 3′UTR in viral host switching and the pathogenicity changes in mice. IMPORTANCE Mosquito-borne flaviviruses (MBFVs) constitute a large number of mosquito-transmitted viruses. The 3′-untranslated region (3′UTR) of MBFV has been suggested to be relevant to viral host-specific adaptation. However, the evolutionary strategies for host-specific fitness among MBFV are different, and the virulence-related structures within the 3′UTR are largely unknown. Here, using Tembusu virus (TMUV), an avian MBFV as models, we observed that the duck-derived SLI of the 3′UTR significantly enhanced the proliferation ability of mosquito-derived TMUV in baby hamster kidney (BHK-21) and duck embryonic fibroblast (DEF) cells, suggesting that the SLI structure was crucial for viral host-specific adaptation of mosquito-derived TMUVs in mammalian and avian cells. In addition, all SLI mutant viruses exhibited reduced viral pathogenicity in mice, indicating that SLI structure was a key factor for the pathogenicity in mice. This study provides a new insight into the functions of the MBFV 3′UTR in viral host switching and pathogenicity changes in mice.

Duck plague virus (DPV) belongs to the alphaherpesvirinae and causes high morbidity and mortality in waterfowl. UL47 is a large abundant structural protein in DPV, which means that UL47 protein plays an important role in virus replication. US3 protein, as a viral protein kinase in alphaherpesviruses, has been reported to be critical for DPV virion assembly. In this study, we over-expressed UL47 and US3 proteins and found that DPV UL47 protein was a phosphorylated substrate of US3 protein, which interacted and co-localized with US3 protein in the cytoplasm. US3-regulated phosphorylation of UL47 was important for the cytoplasmic localization of UL47 because non-phosphorylated UL47 was localized in the nucleus. The six sites of UL47 at Thr29, Ser30, Ser42, Thr47, Ser161, and Thr775 were identified as the phosphorylation targets of US3 protein. In vivo, UL47 phosphorylation was also detected but not in ΔUS3-infected cells. US3 protein promoted the cytoplasmic localization of UL47 at the late stage of infection, and the lack of US3 protein caused a delay in UL47 translocation to the cytoplasm. These results enhance our understanding of the functions of US3 during DPV infection and provide some references for DPV assembly.

Salmonella enterica is a widespread foodborne pathogen with concerning antimicrobial resistance (AMR). Waterfowl are a major source of Salmonella transmission, but there are few systematic studies on Salmonella prevalence in waterfowl species. In this study, 126 Salmonella isolates (65 collected in 2018–2020 and 61 collected in 2002–2005) were obtained from waterfowl samples in Sichuan, China. Their serotypes, pulsed-field gel electrophoresis (PFGE) types, and phenotypic and genotypic AMR profiles were systematically examined. The isolates were distributed in 7 serotypes, including serovars Enteritidis (46.0%), Potsdam (27.8%), Montevideo (7.9%), Cerro (6.3%), Typhimurium (4.8%), Kottbus (4.0%) and Apeyeme (3.2%). Their PFGE characteristics were diverse; all isolates were distributed in four groups (cutoff value: 60.0%) and 20 clusters (cutoff value: 80.0%). Moreover, all isolates were multidrug resistant, and high rates of AMR to lincomycin (100.0%), rifampicin (100.0%), sulfadiazine (93.7%), erythromycin (89.7%), ciprofloxacin (81.0%), and gentamicin (75.4%) were observed. Finally, 49 isolates were subjected to whole-genome sequencing, and a wide variety of AMR genes were found, including multiple efflux pump genes and specific resistance genes. Interestingly, the tet(A)/tet(B) and catII resistance genes were detected in only isolates obtained in the first collection period, while the gyrA (S83F, D87N and D87G) and gyrB (E466D) mutations were detected at higher frequencies in the isolates obtained in the second collection period, supporting the findings that isolates from different periods exhibited different patterns of resistance to tetracycline, chloramphenicol and nalidixic acid. In addition, various incompatible plasmid replicon fragments were detected, including Col440I, Col440II, IncFIB, IncFII, IncX1, IncX9, IncI1-I and IncI2, which may contribute to the horizontal transmission of AMR genes and provide competitive advantages. In summary, we demonstrated that the Salmonella isolates prevalent in Sichuan waterfowl farms exhibited diverse serotypes, multiple AMR phenotypes and genotypes, and AMR changes over time, indicating their potential risks to public health.

ABSTRACT: Flavivirus RNA cap-methylation plays an important role in viral infection, proliferation, and escape from innate immunity. The methyltransferase (MTase) of the flavivirus NS5 protein catalyzes viral RNA methylation. The E218 amino acid of the NS5 protein MTase domain is one of the active sites of flavivirus methyltransferase. In flaviviruses, the E218A mutation abolished 2’-O methylation activity and significantly reduced N-7 methylation activity. Tembusu virus (TMUV, genus Flavivirus) was a pathogen that caused neurological symptoms in ducklings and decreased egg production in laying ducks. In this study, we focused on a comprehensive understanding of the effects of the E218A mutation on TMUV characteristics and the host immune response. E218A mutation reduced TMUV replication and proliferation, but did not affect viral adsorption and entry. Based on a TMUV replicon system, we found that the E218A mutation impaired viral translation. In addition, E218A mutant virus might be more readily recognized by RIG-I-like receptors to activate the corresponding antiviral immune signaling than WT virus. Together, our data suggest that the E218A mutation of TMUV MTase domain impairs viral replication and translation and may activates RIG-I-like receptor signaling, ultimately leading to a reduction in viral proliferation.

ABSTRACT: The nomenclature of duck viral hepatitis (DVH) was historically not a problem. However, 14 hepatotropic viruses among 10 different genera are associated with the same disease name, DVH. Therefore, the disease name increasingly lacks clarity and may no longer fit the scientific description of the disease. Because one disease should not be attributed to 10 genera of viruses, this almost certainly causes misunderstanding regarding the disease-virus relationship. Herein, we revisited the problem and proposed an update to DVH disease classification. This classification is based on the nomenclature of human viral hepatitis and the key principle of Koch's postulates (“one microbe and one disease”). In total, 10 types of disease names have been proposed. These names were literately matched with hepatitis-related viruses. We envision that this intuitive nomenclature system will facilitate scientific communication and consistent interpretation in this field, especially in the Asian veterinary community, where these diseases are most commonly reported.

With the large-scale use of antibiotics, antibiotic resistant bacteria (ARB) continue to rise, and antibiotic resistance genes (ARGs) are regarded as emerging environmental pollutants. The new tetracycline-class antibiotic, tigecycline is the last resort for treating multidrug-resistant (MDR) bacteria. Plasmid-mediated horizontal transfer enables the sharing of genetic information among different bacteria. The tigecycline resistance gene tet(X) threatens the efficacy of tigecycline, and the adjacent ISCR2 or IS26 are often detected upstream and downstream of the tet(X) gene, which may play a crucial driving role in the transmission of the tet(X) gene. Since the first discovery of the plasmid-mediated high-level tigecycline resistance gene tet(X4) in China in 2019, the tet(X) genes, especially tet(X4), have been reported within various reservoirs worldwide, such as ducks, geese, migratory birds, chickens, pigs, cattle, aquatic animals, agricultural field, meat, and humans. Further, our current researches also mentioned viruses as novel environmental reservoirs of antibiotic resistance, which will probably become a focus of studying the transmission of ARGs. Overall, this article mainly aims to discuss the current status of plasmid-mediated transmission of different tet(X) genes, in particular tet(X4), as environmental pollutants, which will risk to public health for the “One Health” concept.

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a diverse family of RNA binding proteins that are implicated in RNA metabolism, such as alternative splicing, mRNA stabilization and translational regulation. According to their different cellular localization, hnRNPs display multiple functions. Most hnRNPs were predominantly located in the nucleus, but some of them could redistribute to the cytoplasm during virus infection. HnRNPs consist of different domains and motifs that enable these proteins to recognize predetermined nucleotide sequences. In the virus-host interactions, hnRNPs specifically bind to viral RNA or proteins. And some of the viral protein-hnRNP interactions require the viral RNA or other host factors as the intermediate. Through various mechanisms, hnRNPs could regulate viral translation, viral genome replication, the switch of translation to replication and virion release. This review highlights the common features and the distinguish roles of hnRNPs in the life cycle of positive single-stranded RNA viruses.

Duck plague caused by duck plague virus (DPV) is a highly contagious disease that can cause serious morbidity and death in waterfowl such as ducks and geese, and bring huge economic losses to the duck industry. In this study, on the basis of the duck plague virus gC gene deletion strain CHv-ΔgC, based on the duck plague virus bacterial artificial chromosome (BAC) platform in our laboratory, the gE gene was knocked out using the traceless deletion technology to obtain gC/gE double gene deletion candidate vaccine strain CHv-ΔgC/gE. The double gene deletion strain (CHv-ΔgC/gE) constructed in this study has greatly weakened virulence, no pathogenicity to ducks, and stable genetic characteristics in vitro and in vivo. Ducks immunized with CHv-ΔgC/gE can produce neutralizing antibodies and ELISA antibody levels comparable to those of commercial duck plague attenuated vaccine immunization, and can resist 100 LD50 CHv challenge of ducks, with good immune protection effect. It has the potential to be further developed into duck plague gC/gE double gene deletion, marked attenuated vaccine.

The viral envelope glycoprotein E (gE) is required for cell-to-cell transmission, anterograde and retrograde neurotransmission, and immune evasion of alphaherpesviruses. gE can also interact with other proteins of the virus and perform various functions in the virus life cycle. In addition, the gE gene is often the target gene for the construction of gene-deleted attenuated marker vaccines. In recent years, new progress has been made in the research and vaccine application of gE with other proteins of the virus. This article reviews the structure of gE, the relationship between gE and other proteins of the virus, and the application of gE in vaccinology, which provides useful information for further research on gE.

Viroporins are virally encoded transmembrane proteins that are essential for viral pathogenicity and can participate in various stages of the viral life cycle, thereby promoting viral proliferation. Viroporins have multifaceted effects on host cell biological functions, including altering cell membrane permeability, triggering inflammasome formation, inducing apoptosis and autophagy, and evading immune responses, thereby ensuring that the virus completes its life cycle. Viroporins are also virulence factors, and their complete or partial deletion often reduces virion release and reduces viral pathogenicity, highlighting the important role of these proteins in the viral life cycle. Thus, viroporins represent a common drug-protein target for inhibiting drugs and the development of antiviral therapies. This article reviews current studies on the functions of viroporins in the viral life cycle and their regulation of host cell responses, with the aim of improving the understanding of this growing family of viral proteins.