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

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

Specific DNAzymes cleave the 300-618 nt of 5'UTR to inhibit DHAV-1 translation and replication.

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.

Duck plague virus US3 protein kinase phosphorylates UL47 and regulates the subcellular localization of UL47.

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.

Evaluation of safety and immunogenicity of duck-plague virus gC/gE double gene deletion.

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 DHAV-1 protein VP1 interacts with PI3KC3 to induce autophagy through the PI3KC3 complex.

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.

Duck Plague Virus Negatively Regulates IFN Signaling to Promote Virus Proliferation via JNK Signaling Pathway.

Duck plague virus (DPV), a member of the alphaherpesvirus subfamily, can cause severe damage and immunosuppression in ducks and geese in China. Since lacking an available cell model, the antiviral signal transduction pathways induction and regulation mechanisms related to DPV infection in duck cells are still enigmatic. Our previous study developed a monocyte/macrophages cell model, which has been applied to study innate immunity with DPV. In the present study, we compared and analyzed transcriptome associated with the DPV infection of CHv (virulent strain) and CHa (avirulent strain) at 48hpi based on the duck monocyte/macrophages cell model and RNA-seq technology. Differentially expressed genes (DEGs) analysis showed 2,909 and 2,438 genes altered in CHv and CHa infected cells compared with control cells. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in biological processes such as metabolic pathways, viral infectious diseases, immune system, and signal transduction. The CHv and CHa virus differentially regulated MAPK, NF-κB, and IFN signaling pathways based on transcriptome sequencing data and RT-qPCR results. The JNK inhibitor SP600125 enhanced the IFN signaling, but potentially reduced the VSV and DPV titers in the cell culture supernatant, indicating that JNK negatively regulates the IFN pathway and the inflammatory pathway to promote virus proliferation. The research results may provide promising information to understand the pathogenesis of DPV and provide a novel mechanism by which DPV modulates antiviral signaling and facilitate virus proliferation through hijacking the JNK pathway, which provides a new means for the prevention and control of DPV infection.

Identification of duck GSDME: Tissue distribution, proteolysis and cellular location.

Gasdermin E (GSDME) is a member of the gasdermin family. Cleavage of mammalian GSDME by apoptotic caspases or granzyme proteases liberates the N-terminal effector domain (GSDME-N), which is capable of forming membrane pores and executing inflammation and cell death. Herein, duck GSDME was first cloned with a total length of 1500 bp and encoding 499 amino acids (aa), which is most evolutionally related to the chicken GSDME. The tissue-distribution profiles of GSDME showed that relatively high levels of GSDME mRNA were detected in immune tissues of duckling and adult ducks. Additionally, GSDME mRNA was significantly upregulated in duck primary embryo fibroblasts (DEFs) and duck primary ovary cells after duck Tembusu virus (DTMUV) infection. Intriguingly, when duck caspase-3 was coexpressed, the duck GSDME produced two GSDME-N fragments with molecular weights of 25 kDa and 30 kDa. Furthermore, both GSDME and cleaved GSDME were observed to be located in the cytoplasm by indirect immunofluorescence assay (IFA). Taken together, our research data show that duck GSDME has similar biological characteristics to mammals. These findings highlight the role of duck GSDME in TMUV infection, indicating that cooperation between GSDME and caspase-3 promotes the proteolytic process.

The lysine at position 151 of the duck hepatitis A virus 1 2C protein is critical for its NTPase activities.

The duck hepatitis A virus 1 (DHAV-1) 2C protein was predicted to be a superfamily III helicase member and includes nucleotide binding (NTB) and putative RNA helicase activity motifs. To study whether DHAV-1 2C protein has NTB activity, we expressed DHAV-1 2C protein with maltose binding protein (MBP) to solve its poor solubility in a prokaryotic expression system. We showed that the DHAV-1 2C protein has nucleoside triphosphatase (NTPase) activity by measuring the released phosphate. The NTPase of the DHAV-1 2C protein is Mg2+ indispensable and affected by other biochemical characteristics such as Mn2+, Ca2+, Zn2+, Na+ and pH. Guanidine hydrochloride (GdnHCl), a potent inhibitor of viral RNA replication, inhibited ATPase activity of the DHAV-1 2C protein in a dose-dependent manner. Finally, we constructed three mutants to identify the key site for the ATPase activity of the DHAV-1 2C protein. These results indicate that lysine at position 151 of the DHAV-1 2C protein is very important for NTPase activity. Here, we demonstrated and partially characterized that the DHAV-1 2C protein has NTPase activity and showed that mutation of the lysine in the conserved Walker A impairs that activity. The results serve to confirm what is readily predicted from previous work on picornavirus 2C proteins. It also provides a basis for further study of the 2C protein and the function of NTPase activity on the viral life cycle.

Decreased virulence of duck Tembusu virus harboring a mutant NS2A with impaired interaction with STING and IFNß induction.

Our previous studies revealed that duck Tembusu virus (DTMUV) NS2A inhibited IFNß signaling pathway by competitively binding to STING with TBK1, leading to reducing the phosphorylation of TBK1. Herein, we found that the 114-143 aa region of NS2A is critical for its interaction with STING and suppression of STING-mediated IFNß signaling. We further identified the amino acids at positions L129, N130, L139, R140 and F143 of NS2A critical for NS2A-STING interaction. Subsequently, single residue substitution in the NS2A protein was introduced into the DTMUV replicon and infectious clone. The replicons with NS2A L129A and L130A mutations significantly inhibited viral genome RNA replication. The rDTMUV NS2A L129A, L139A and R140A mutant viruses yielded significantly lower titer levels than WT in both BHK-21 and DEF cells, with much more obvious effect on the viral genome level, and infectious virions formed outside of infected cells. Especially, the rDTMUV L129A mutant showed a significantly lower mortality in both embryos and ducks than WT. All NS2A-mutants decreased the weight gain of infected ducklings and reduced the viral loads in the spleen relative to WT. However, no significant differences of viral loads were observed in the blood, thymus, or liver. Our findings extend our previous study on the immune evasion role of flavivirus NS2A protein. The targeted therapy of disabling the viral strategies developed for evading innate defense can be applied to the development of attenuated flaviviruses.

DHAV-1 Blocks the Signaling Pathway Upstream of Type I Interferon by Inhibiting the Interferon Regulatory Factor 7 Protein.

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)-ß 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-ß-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.

The LORF5 Gene Is Non-essential for Replication but Important for Duck Plague Virus Cell-to-Cell Spread Efficiently in Host Cells.

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.

Amelioration of Beta Interferon Inhibition by NS4B Contributes to Attenuating Tembusu Virus Virulence in Ducks.

Our previous studies reported that duck Tembusu virus nonstructural protein 2A (NS2A) is a major inhibitor of the IFNß signaling pathway through competitively binding to STING with TBK1, leading to a reduction in TBK1 phosphorylation. Duck TMUV NS2B3 could cleave and bind STING to subvert the IFNß signaling pathway. Here, we found that overexpression of duck TMUV NS4B could compete with TBK1 in binding to STING, reducing TBK1 phosphorylation and inhibiting the IFNß signaling pathway by using the Dual-Glo® Luciferase Assay System and the NanoBiT protein-protein interaction (PPI) assay. We further identified the E2, M3, G4, W5, K10 and D34 residues in NS4B that were important for its interaction with STING and its inhibition of IFNß induction, which were subsequently introduced into a duck TMUV replicon and an infectious cDNA clone. We found that the NS4B M3A mutant enhanced RNA replication and exhibited significantly higher titer levels than WT at 48-72 hpi but significantly decreased mortality (80%) in duck embryos compared to WT (100%); the NS4B G4A and R36A mutants slightly reduced RNA replication but exhibited the same titer levels as WT. However, the NS4B R36A mutant did not attenuate the virulence in duck embryos, whereas the G4A mutant significantly decreased the mortality (70%) of duck embryos. In addition, the NS4B W5A mutant did not affect viral replication, whereas the D34A mutant slightly reduced RNA replication, and both mutants exhibited significantly lower titer levels than the WT and significantly decreased mortality (90% and 70%, respectively) in duck embryos. Hence, our findings provide new insight into the development of attenuated flaviviruses by targeting the disabling viral strategies used to evade the innate defense mechanisms.

Emergence of a novel pegivirus species in southwest China showing a high rate of coinfection with parvovirus and circovirus in geese.

Previously, we isolated a novel strain of goose pegivirus (GPgV) that infects geese and shows high levels of lymphotropism. This novel pegivirus strain is phylogenetically distinct from previously known Pegivirus species, Pegivirus A-K, and qualifies as a candidate new Pegivirus species, GPgV. GPgV is tentatively named Pegivirus M. Here, to better understand the epidemic of GPgV infection and the coinfection of this virus with other viruses in Southwest China, 25 geese in poor health from Sichuan Province and 24 geese in poor health from the municipality of Chongqing were collected. The geese were tested for 9 types of goose viruses (goose hemorrhagic polyomavirus, GPgV, astrovirus, parvovirus, circovirus, reovirus, coronavirus, paramyxovirus, and avian influenza virus) by RT-PCR or nested RT-PCR. GPgV RNA was detected in 2 out of 25 monoinfections and 8 out of 25 coinfections with other viruses on Sichuan farms and 2 out of 24 monoinfections and 10 out of 24 coinfections on Chongqing farms. Overall, 22 of the 49 (44.9%) geese were positive for GPgV, which indicated a high infection rate. To the best of our knowledge, this is the first report of GPgV coinfection with other epidemic viruses. This study enhances our understanding of the emergence and epidemiology of Pegivirus.

Molecular cloning of duck CD40 and its immune function research.

Cosignal molecules are cell surface molecules that transduce signals to other cells to modulate immune response positively (costimulate) or negatively (cosuppress). Costimulatory signals are key factors in determining whether T/B cells are capable of responding to specific antigens and ultimately mediating an appropriate immune response. In this study, the cDNA sequence containing the complete coding frame of the costimulatory molecule duck CD40 gene was cloned and reported for the first time, and its mediated antiviral innate immune was verified in vitro. Results suggested duck CD40 molecule plays an important role in the innate immune responsiveness against some viruses. These data will be beneficial for the further understand of the avian immune system.

Duck Hepatitis A Virus Type 1 Induces eIF2α Phosphorylation-Dependent Cellular Translation Shutoff via PERK/GCN2.

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.

SC75741 antagonizes vesicular stomatitis virus, duck Tembusu virus, and duck plague virus infection in duck cells through promoting innate immune responses.

Duck Tembusu virus (DTMUV) and duck plague virus (DPV) are typical DNA and RNA viruses of waterfowl, causing drastic economic losses to the duck farm industry in terms of high mortality and decreased egg production. These 2 viruses reappear from time to time because the available vaccines fail to provide complete immunity and no clinical antiviral drugs are available for them. In the present study, we evaluated the antiviral activity of SC75741 for DTMUV, DPV, and the model virus, vesicular stomatitis virus infection in duck cells. SC75741, a nuclear factor-kappa B (NF-κB)-specific inhibitor in mammal cells, revealed the highest antiviral activity among the inhibitors specific to c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase (p38), and NF-κB signaling. The antiviral activity of SC75741 was dose-dependent and showed effects in different duck cell types. Time-addition and duration assay demonstrated that SC75741 inhibited virus infection in the middle of and after virus infection at least for 72 h in duck embro fibroblast cells. The DPV viral adsorption and genomic copy number were reduced, indicating that SC75741 blocks the phase of the virus life cycle at viral entry and genomic replication. In addition, SC75741 enhanced the expression of interferon only when stimulator of interferon genes (STING) was overexpressed or pre-activated by the virus infection, suggesting that SC75741 acts as a STING agonist. In conclusion, SC75741 is a candidate antiviral agent for DTMUV and DPV.

Corrigendum: Autophagy Is a Potential Therapeutic Target Against Duck Tembusu Virus Infection in Vivo.

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The lipopolysaccharide outer core transferase genes pcgD and hptE contribute differently to the virulence of Pasteurella multocida in ducks.

Fowl cholera caused by Pasteurella multocida exerts a massive economic burden on the poultry industry. Lipopolysaccharide (LPS) is essential for the growth of P. multocida genotype L1 strains in chickens and specific truncations to the full length LPS structure can attenuate bacterial virulence. Here we further dissected the roles of the outer core transferase genes pcgD and hptE in bacterial resistance to duck serum, outer membrane permeability and virulence in ducks. Two P. multocida mutants, ΔpcgD and ΔhptE, were constructed, and silver staining confirmed that they all produced truncated LPS profiles. Inactivation of pcgD or hptE did not affect bacterial susceptibility to duck serum and outer membrane permeability but resulted in attenuated virulence in ducks to some extent. After high-dose inoculation, ΔpcgD showed remarkably reduced colonization levels in the blood and spleen but not in the lung and liver and caused decreased injuries in the spleen and liver compared with the wild-type strain. In contrast, the ΔhptE loads declined only in the blood, and ΔhptE infection caused decreased splenic lesions but also induced severe hepatic lesions. Furthermore, compared with the wild-type strain, ΔpcgD was significantly attenuated upon oral or intramuscular challenge, whereas ΔhptE exhibited reduced virulence only upon oral infection. Therefore, the pcgD deletion caused greater virulence attenuation in ducks, indicating the critical role of pcgD in P. multocida infection establishment and survival.

Comparative genomics and metabolomics analysis of Riemerella anatipestifer strain CH-1 and CH-2.

Riemerella anatipestifer is a major pathogenic microorganism in poultry causing serositis with significant mortality. Serotype 1 and 2 were most pathogenic, prevalent, and liable over the world. In this study, the intracellular metabolites in R. anatipestifer strains RA-CH-1 (serotype 1) and RA-CH-2 (serotype 2) were identified by gas chromatography-mass spectrometer (GC-MS). The metabolic profiles were performed using hierarchical clustering and partial least squares discriminant analysis (PLS-DA). The results of hierarchical cluster analysis showed that the amounts of the detected metabolites were more abundant in RA-CH-2. RA-CH-1 and RA-CH-2 were separated by the PLS-DA model. 24 potential biomarkers participated in nine metabolisms were contributed predominantly to the separation. Based on the complete genome sequence database and metabolite data, the first large-scale metabolic models of iJL463 (RA-CH-1) and iDZ470 (RA-CH-2) were reconstructed. In addition, we explained the change of purine metabolism combined with the transcriptome and metabolomics data. The study showed that it is possible to detect and differentiate between these two organisms based on their intracellular metabolites using GC-MS. The present research fills a gap in the metabolomics characteristics of R. anatipestifer.