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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 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 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.

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 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.

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 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 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.

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 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.

The herpesvirus UL11 protein is encoded by the UL11 gene and is a membrane-anchored protein with multiple functions. In the last stage of viral replication, UL11 participates in the secondary envelopment process. It also plays a key role in primary envelopment, the transportation of newly assembled viral particles through cytoplasmic vesicles, and virion egress from the cell. UL11 is an important accessory protein and sometimes cooperates with other proteins that participate in virus-induced cell fusion. Cell fusion is necessary for cell-to-cell transmissions. This review summarizes the latest literature and discusses the roles of UL11 in viral assembly, primary and secondary envelopment, and cell-to-cell transmission to obtain a better understanding of the UL11 protein in the life cycle of herpesviruses and to serve as a reference for studying other viruses. Additionally, some recently discovered characteristics of UL11 are summarized.

Tegument protein UL11 plays a critical role in the life cycle of herpesviruses. The UL11 protein of herpesviruses is important for viral particle entry, release, assembly, and secondary envelopment. Lipid raft is cholesterol-rich functional microdomains in cell membranes, which plays an important role in signal transduction and substance transport. Flotillin and prohibition, which are considered to be specific markers of lipid raft. However, little is known about the function of duck plague virus (DPV) UL11 in the life cycle of the viruses and the relationship between the lipid raft and UL11. In this study, an interference plasmid shRNA126 for UL11 was used. Results showed that UL11 is involved in the replication, cell to cell spread, viral particle assembly, and release processes. Furthermore, UL11 was verified that it could interact with the lipid raft through sucrose density gradient centrifugation and that function correlates with the second glycine of the UL11. When the lipid raft was depleted using the methyl-β-cyclodextrin, the release of the DPV was decreased. Moreover, UL11 can decrease several relative viral genes mRNA levels by qRT-PCR and Western blot test. Altogether, these results highlight an important role for UL11 protein in the viral replication cycle.

Duck plague virus (DPV) can cause high morbidity and mortality in many waterfowl species within the order Anseriformes. The DPV genome contains 78 open reading frames (ORFs), among which the LORF2, LORF3, LORF4, LORF5, and SORF3 genes are unique genes of avian herpesvirus. In this study, to investigate the role of this unique LORF5 gene in DPV proliferation, we generated a recombinant virus that lacks the LORF5 gene by a two-step red recombination system, which cloned the DPV Chinese virulent strain (DPV CHv) genome into a bacterial artificial chromosome (DPV CHv-BAC); the proliferation law of LORF5-deleted mutant virus on DEF cells and the effect of LORF5 gene on the life cycle stages of DPV compared with the parent strain were tested. Our data revealed that the LORF5 gene contributes to the cell-to-cell transmission of DPV but is not relevant to virus invasion, replication, assembly, and release formation. Taken together, this study sheds light on the role of the avian herpesvirus-specific gene LORF5 in the DPV proliferation life cycle. These findings lay the foundation for in-depth functional studies of the LORF5 gene in DPV or other avian herpesviruses.

Duck plague caused by the duck plague virus (DPV) is an infectious disease that seriously harms the waterfowl breeding industry. The VP16 protein of α herpesvirus can bind to specific cis-acting elements upstream of the promoter of the immediate-early (IE, α) gene to promote the transcription of the IE gene, so it is also called the trans-inducer of IE gene (α-TIF). However, no studies on DPV α-TIF have been reported. This study investigated the DPV pUL48, a homolog of HSV-1 VP16, transcriptional activation region, target sequence, and viral protein affecting its transcriptional activation using a dual-luciferase reporter gene detection system, and pUL48 was identified as the α-TIF of DPV. (1) The regulation of pUL48 on DPV different gene promoters showed that pUL48 could activate all the promoters of IE genes (ICP4, ICP22, and ICP27) but not the promoters of early and late genes. (2) The activity of pUL48 to ICP4 and ICP22 promoters with different upstream lengths showed that pUL48 activated ICP4 and ICP22 promoters by acting on TAATGA (T) TAT element upstream of ICP4 promoter and TAATTATAT element upstream of ICP22 promoter, respectively. (3) Transcriptional activation of IE gene by truncated proteins of different lengths at the N-terminal of pUL48 was detected. The results showed that the transcriptional activation domain of pUL48 was amino acids 1–60 at the N-terminal, and amino acids 1–20 was its core region. In addition, it was found that pUL14, pUL46, and pUL47 significantly promoted the transcriptional activation of pUL48. The effects of loss of pUL47 and its nuclear localization signal on the nuclear entry and transcriptional activation function of pUL48 were further examined. The results showed that pUL47 could promote the nuclear entry of pUL48 through its nuclear localization signal at positions 40–50 and 768–777 amino acids, thus, enhancing the transcriptional activation function of pUL48 and synergistic promotion of viral gene transcription.

Duck hepatitis A virus (DHAV), which mainly infects 1- to 4-week-old ducklings, has a fatality rate of 95% and poses a huge economic threat to the duck industry. However, the mechanism by which DHAV-1 regulates the immune response of host cells is rarely reported. This study examined whether DHAV-1 contains a viral protein that can regulate the innate immunity of host cells and its specific regulatory mechanism, further exploring the mechanism by which DHAV-1 resists the host immune response. In the study, the dual-luciferase reporter gene system was used to screen the viral protein that regulates the host innate immunity and the target of this viral protein. The results indicate that the DHAV-1 3C protein inhibits the pathway upstream of interferon (IFN)-β by targeting the interferon regulatory factor 7 (IRF7) protein. In addition, we found that the 3C protein inhibits the nuclear translocation of the IRF7 protein. Further experiments showed that the 3C protein interacts with the IRF7 protein through its N-terminus and that the 3C protein degrades the IRF7 protein in a caspase 3-dependent manner, thereby inhibiting the IFN-β-mediated antiviral response to promote the replication of DHAV-1. The results of this study are expected to serve as a reference for elucidating the mechanisms of DHAV-1 infection and pathogenicity.

In our previous study, it was shown that Riemerella anatipestifer, a Gram-negative bacterium, is naturally competent, but the genes involved in the process of natural transformation remain largely unknown. In this study, a random transposon mutant library was constructed using the R. anatipestifer ATCC11845 strain to screen for the genes involved in natural transformation. Among the 3000 insertion mutants, nine mutants had completely lost the ability of natural transformation, and 14 mutants showed a significant decrease in natural transformation frequency. We found that the genes RA0C_RS04920, RA0C_RS04915, RA0C_RS02645, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, RA0C_RS09020, and RA0C_RS04870 are essential for the occurrence of natural transformation in R. anatipestifer ATCC11845. In particular, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, and RA0C_RS04870 were putatively annotated as ComEC, DprA, ComF, and RecA proteins, respectively, in the NCBI database. However, RA0C_RS02645, RA0C_RS04920, RA0C_RS04915, and RA0C_RS09020 were annotated as proteins with unknown function, with no homology to any well-characterized natural transformation machinery proteins. The homologs of these proteins are mainly distributed in the members of Flavobacteriaceae. Taken together, our results suggest that R. anatipestifer encodes a unique natural transformation machinery.

Riemerella anatipestifer causes serious contagious disease in ducks, geese, and other fowl. However, as a harmful pathogen causing significant economic losses in the poultry industry, R. anatipestifer is still poorly understood for its pathogenesis mechanisms. In a previous study, we developed an indirect ELISA method for detecting R. anatipestifer infection using B739_0832 protein, a putative outer membrane protein H (OmpH) that is conserved among different serotypes of R. anatipestifer. Although OmpH in some pathogenic bacteria, such as Pasteurella, has been reported as a virulence factor, it is still not clear whether B739_0832 protein contributes to the virulence of R. anatipestifer. In this study, we confirmed that B739_0832 protein in R. anatipestifer localizes to the outer membrane. We constructed a B739_0832 deletion mutant strain (ΔB739_0832) and assayed various effects from the deletion of B739_0832. ΔB739_0832 strain had a similar growth rate to wild-type R. anatipestifer CH-1. However, the survival rate of ducklings in 10 days after infection from ΔB739_0832 strain was 50%, whereas no ducklings survived from wild-type R. anatipestifer infection. Furthermore, the median lethal dose (LD50) of the ΔB739_0832 strain was approximately 150 times higher than that of the wild-type strain. Pathology examinations on infected ducklings found that, at 36 h after infection, bacterial loads in blood, liver, and brain tissues from ΔB739_0832-infected ducklings were considerably lower than those from wild-type infected ducklings. These results demonstrate that the B739_0832 protein contributes to the virulence of R. anatipestifer CH-1.

Riemerella anatipestifer is a gram-negative bacterium that is the first naturally competent bacterium identified in the family Flavobacteriaceae. However, the determinants that influence the natural transformation and the underlying mechanism remain unknown. In this study, we evaluated the effects of various nutritional factors of the GCB medium [glucose, L-glutamine, vitamin B1, Fe (NO3)3, NaCl, phosphate, and peptone], on the natural transformation of R. anatipestifer ATCC 11845. Among the assayed nutrients, peptone and phosphate affected the natural transformation of R. anatipestifer ATCC 11845, and the transformation frequency was significantly decreased when phosphate or peptone was removed from the GCB medium. When the iron chelator 2,2′-dipyridyl (Dip) was added, the transformation frequency was decreased by approximately 100-fold and restored gradually when Fe (NO3)3 was added, suggesting that the natural transformation of R. anatipestifer ATCC 11845 requires iron. Given the importance of TonB in nutrient transportation, we further identified whether TonB is involved in the natural transformation of R. anatipestifer ATCC 11845. Mutation of tonBA or tonBB, but not tbfA, was shown to inhibit the natural transformation of R. anatipestifer ATCC 11845 in the GCB medium. In parallel, it was shown that the tonBB mutant, but not the tonBA mutant, decreased iron acquisition in the GCB medium. This result suggested that the tonBB mutant affects the natural transformation frequency due to the deficiency of iron utilization.

Herpesviruses are extremely successful parasites that have evolved over millions of years to develop a variety of mechanisms to coexist with their hosts and to maintain host-to-host transmission and lifelong infection by regulating their life cycles. The life cycle of herpesviruses consists of two phases: lytic infection and latent infection. During lytic infection, active replication and the production of numerous progeny virions occur. Subsequent suppression of the host immune response leads to a lifetime latent infection of the host. During latent infection, the viral genome remains in an inactive state in the host cell to avoid host immune surveillance, but the virus can be reactivated and reenter the lytic cycle. The balance between these two phases of the herpesvirus life cycle is controlled by broad interactions among numerous viral and cellular factors. ICP22/ORF63 proteins are among these factors and are involved in transcription, nuclear budding, latency establishment, and reactivation. In this review, we summarized the various roles and complex mechanisms by which ICP22/ORF63 proteins regulate the life cycle of human herpesviruses and the complex relationships among host and viral factors. Elucidating the role and mechanism of ICP22/ORF63 in virus–host interactions will deepen our understanding of the viral life cycle. In addition, it will also help us to understand the pathogenesis of herpesvirus infections and provide new strategies for combating these infections.

Flavivirus envelope protein (E) plays an important role in cellular infection, especially in virulence and antigenicity. E domain III of Tembusu virus (TMUV) is highly conserved among flaviviruses and contains four loop regions. However, the functions of the loop regions of TMUV E domain III in the viral life cycle have not yet been discovered. In this study, using a reverse genetics system, we performed site-directed mutagenesis on loops I, II, III, and IV of TMUV E domain III. Mutant 6 (S388A.G389A.K390A) showed better proliferation than the wild-type virus, while mutants 1–5 exhibited decreased in vitro infectivity, as determined by immunofluorescence assay (IFA). Based on a TMUV replicon system, the mutations exhibited no apparent effect on TMUV RNA replication. Subcellular fractionation assays and packaging system assays indicated that mutations in loops II–IV (T332A, T332S, S365A.S366A.T367A, and S388A.G389A.K390A, respectively) disrupted virion assembly. Moreover, loops I–IV played an important role in virus binding and entry, while mutant 6 (S388A.G389A.K390A) exhibited robust activity in virus entry. Taken together, our findings indicated the critical role of the loop regions in TMUV E domain III in the virus entry and assembly process.

Duck hepatitis A virus type 1 (DHAV-1) is one of the most deadly pathogens that endanger the duck industry. Most viruses usually turn off host translation after infection to facilitate viral replication and translation. For the first time report to our knowledge, DHAV-1 can induce eIF2α phosphorylation and inhibit cellular translation in duck embryo fibroblasts (DEFs). Moreover, the activity of DHAV-1 in the cells caused obvious eIF2α phosphorylation, which has nothing to do with the viral protein. Subsequently, we screened two kinases (PERK and GCN2) that affect eIF2α phosphorylation through inhibitors and shRNA. Notably, the role of GCN2 in other picornaviruses has not been reported. In addition, when the phosphorylation of eIF2α induced by DHAV-1 is inhibited, the translation efficiency of DEFs restores to a normal level, indicating that DHAV-1 induced cellular translation shutoff is dependent on eIF2α phosphorylation.

In a previous study, it was shown that Riemerella anatipestifer, a member of Flavobacteriaceae, is naturally competent. However, whether natural competence is universal in Flavobacteriaceae remains unknown. In this study, it was shown for the first time that Riemerella columbina was naturally competent in the laboratory condition; however, Flavobacterium johnsoniae was not naturally competent under the same conditions. The competence of R. columbina was maintained throughout the growth phases, and the transformation frequency was highest during the logarithmic phase. A competition assay revealed that R. columbina preferentially took up its own genomic DNA over heterologous DNA. The natural transformation frequency of R. columbina was significantly increased in GCB medium without peptone or phosphate. Furthermore, natural transformation of R. columbina was inhibited by 0.5 mM EDTA, but could be restored by the addition of CaCl2, MgCl2, ZnCl2, and MnCl2, suggesting that these divalent cations promote the natural transformation of R. columbina. Overall, this study revealed that natural competence is not universal in Flavobacteriaceae members and triggering of competence differs from species to species.

The envelope glycoprotein M (gM), a surface virion component conserved among alphaherpesviruses, is a multiple-transmembrane domain-containing glycoprotein with a complex N-linked oligosaccharide. The gM mediates a diverse range of functions during the viral life cycle. In this review, we summarize the biological features of gM, including its characterization and function in some specicial alphaherpesviruses. gM modulates the virus-induced membrane fusion during virus invasion, transports other proteins to the appropriate intracellular membranes for primary and secondary envelopment during virion assembly, and promotes egress of the virus. The gM can interact with various viral and cellular components, and the focus of recent research has also been on interactions related to gM. And we will discuss how gM participates in the life cycle of alphaherpesviruses.

The protein encoded by the UL48 gene of alphaherpesviruses is named VP16 or alpha-gene-transactivating factor (α-TIF). In the early stage of viral replication, VP16 is an important transactivator that can activate the transcription of viral immediate-early genes, and in the late stage of viral replication, VP16, as a tegument, is involved in viral assembly. This review will explain the mechanism of VP16 acting as α-TIF to activate the transcription of viral immediate-early genes, its role in the transition from viral latency to reactivation, and its effects on viral assembly and maturation. In addition, this review also provides new insights for further research on the life cycle of alphaherpesviruses and the role of VP16 in the viral life cycle.

The 3′ untranslated region (3′ UTR) of positive-sense single-stranded RNA [ssRNA(+)] viruses is highly structured. Multiple elements in the region interact with other nucleotides and proteins of viral and cellular origin to regulate various aspects of the virus life cycle such as replication, translation, and the host-cell response. This review attempts to summarize the primary and higher order structures identified in the 3′UTR of ssRNA(+) viruses and their functional roles.

Enteroviral replication reorganizes the cellular membrane. Upon infection, viral proteins and hijacked host factors generate unique structures called replication organelles (ROs) to replicate their viral genomes. ROs promote efficient viral genome replication, coordinate the steps of the viral replication cycle, and protect viral RNA from host immune responses. More recent researches have focused on the ultrastructure structures, formation mechanism, and functions in the virus life cycle of ROs. Dynamic model of enterovirus ROs structure is proposed, and the secretory pathway, the autophagy pathway, and lipid metabolism are found to be associated in the formation of ROs. With deeper understanding of ROs, some compounds have been found to show inhibitory effects on viral replication by targeting key proteins in the process of ROs formation. Here, we review the recent findings concerning the role, morphology, biogenesis, formation mechanism, and inhibitors of enterovirus ROs.

Alphaherpesviruses are zoonotic pathogens that can cause a variety of diseases in humans and animals and severely damage health. Alphaherpesvirus infection is a slow and orderly process that can lie dormant for the lifetime of the host but may be reactivated when the immune system is compromised. All alphaherpesviruses feature a protein layer called the tegument that lies between the capsid and the envelope. Virus protein (VP) 22 is one of the most highly expressed tegument proteins; there are more than 2,000 copies of this protein in each viral particle. VP22 can interact with viral proteins, cellular proteins, and chromatin, and these interactions play important roles. This review summarizes the latest literature and discusses the roles of VP22 in viral gene transcription, protein synthesis, virion assembly, and viral cell-to-cell spread with the purpose of enhancing understanding of the life cycle of herpesviruses and other pathogens in host cells. The molecular interaction information herein provides important reference data.

Enterovirus infection can cause a variety of diseases and severely impair the health of humans, animals, poultry, and other organisms. To resist viral infection, host organisms clear infected cells and viruses via apoptosis. However, throughout their long-term competition with host cells, enteroviruses have evolved a series of mechanisms to regulate the balance of apoptosis in order to replicate and proliferate. In the early stage of infection, enteroviruses mainly inhibit apoptosis by regulating the PI3K/Akt pathway and the autophagy pathway and by impairing cell sensors, thereby delaying viral replication. In the late stage of infection, enteroviruses mainly regulate apoptotic pathways and the host translation process via various viral proteins, ultimately inducing apoptosis. This paper discusses the means by which these two phenomena are balanced in enteroviruses to produce virus-favoring conditions – in a temporal sequence or through competition with each other. This information is important for further elucidation of the relevant mechanisms of acute infection by enteroviruses and other members of the picornavirus family.

Duck tembusu virus (DTMUV) is newly emerged in poultry and causes great losses to the breeding industry in China and neighboring countries. Effective antiviral strategies are still being studied. Autophagy is a cellular degradative pathway, and our lab's previous data show that autophagy promotes DTMUV replication in vitro. To study the role of autophagy further in vivo, we utilized ducks as the animal model to investigate the autophagy responses in DTMUV-targeted tissues. And also, we utilized autophagy regulators, including Rapamycin (Rapa) as the autophagy enhancer, 3-Methyladenine (3-MA) and Chloroquine (CQ) as the autophagy inhibitors, to adjust the host autophagic levels and then study the effects of autophagy on tissue damages and virus replication. As a result, we first found DTMUV infection trigged autophagy and autophagy regulator treatments regulated autophagy levels successfully in duck spleens and brains. Next, we found that autophagy inhibitors inhibited DTMUV replication and alleviated DTMUV-induced pathological symptoms, whereas the autophagy inducer treatment led to the opposite effects. And we also found that autophagic regulation was correlated with the expression of innate immune genes, including pattern recognition receptors, type I interferons, and cytokines, and caused different effects in different tissues. In summary, we demonstrated that autophagy facilitated DTMUV replication, aggravated the developments of pathological symptoms and possibly counteracts the host's innate immunity response in vivo.

The duplicate US1 genes of duck enteritis virus (DEV) encode a protein with a conserved Herpes_IE68 domain, which was found to be closely related to the herpes virus immediate early regulatory protein family and is highly conserved among counterparts encoded by Herpes_IE68 genes. Previous studies found the homologous proteins HSV-1 ICP22 and VZV ORF63/ORF70 to be critical for virus transcription and replication. However, little is known about the DEV ICP22 protein. In this paper, we describe the characteristics of this protein based on pharmacological experiments, real-time quantitative Polymerase Chain Reaction, Western blot, and immunofluorescence assays. We also investigate the role of the protein in DEV replication via mutation of US1. As a result, we found that the DEV ICP22 protein is a non-essential immediate early protein predominantly located in the nucleus of infected DEF cells and that DEV replication is impaired by US1 deletion. We also found that ICP22 contains a classical nuclear localization signal (NLS) at 305-312AA, and ICP22 cannot enter the nucleus by itself after mutating residue 309.

Cell death is a fundamental process in maintaining cellular homeostasis, which can be either accidental or programed. Programed cell death depends on the specific signaling pathways, resulting in either lytic or non-lytic morphology. It exists in two primary forms: apoptosis and autophagic cell death. Apoptosis is a non-lytic and selective cell death program, which is executed by caspases in response to non-self or external stimuli. In contrast, autophagy is crucial for maintaining cellular homeostasis via the degradation and recycling of cellular components. These two mechanisms also function in the defense against pathogen attack. However, picornaviruses have evolved to utilize diverse strategies and target critical components to regulate the apoptotic and autophagic processes for optimal replication and the release from the host cell. Although an increasing number of investigations have shown that the apoptosis and autophagy are altered in picornavirus infection, the mechanism by which viruses take advantage of these two processes remains unknown. In this review, we discuss the mechanisms of picornavirus executes cellular apoptosis and autophagy at the molecular level and the relationship between these interactions and viral pathogenesis.

Live attenuated vaccines are widely used to protect humans or animals from pathogen infections. We have previously developed a chicken embryo-attenuated Duck Hepatitis A Virus genotype 1 (DHAV-1) vaccine (CH60 strain). This study aims to understand the mechanisms that drive a virulent strain to an attenuated virus. Here, we systematically compared five DHAV-1 chicken embryo attenuated strains and 68 virulent strains. Phylogenetic analysis indicated that duck virulent strains isolated from different geographic regions of China undergo a convergent evolution in the chicken embryos. Comparative analysis indicated that the codon usage bias of the attenuated strains were shaped by chicken codons usage bias, which essentially contributed to viral adaption in the unsuitable host driven by incompatible translation. Of note, the missense mutations in coding region and mutations in untranslated regions may also contribute to viral attenuation of DHAV-1 to some extent. Importantly, we have experimentally confirmed that the expression levels of four viral proteins (2A3pro, 2A3pro, 3Cpro, and 3Dpro) in the liver and kidney of ducks infected with an attenuated strain are significantly lower than that infected with a virulent strain, despite with similar virus load. Thus, the key mechanisms of viral attenuation revealed by this study may lead to innovative and easy approaches in designing live attenuated vaccines.