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.

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 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.

The First Nonmammalian Pegivirus Demonstrates Efficient In Vitro Replication and High Lymphotropism.

Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, including nonhuman primates, bats, horses, pigs, and rodents, but are not associated with disease. Here, we report a new, genetically distinct pegivirus in goose (Anser cygnoides), the first identified in a nonmammalian host species. Goose pegivirus (GPgV) can be propagated in goslings, embryonated goose eggs, and primary goose embryo fibroblasts, and is thus the first pegivirus that can be efficiently cultured in vitro Experimental infection of GPgV in goslings via intravenous injection revealed robust replication and high lymphotropism. Analysis of the tissue tropism of GPgV revealed that the spleen and thymus were the organs bearing the highest viral loads. Importantly, GPgV could promote clinical manifestations of goose parvovirus infection, including reduced weight gain and 7% mortality. This finding contrasts with the lack of pathogenicity that is characteristic of previously reported pegiviruses.IMPORTANCE Members of the Pegivirus genus, family Flaviviridae, widely infect humans and other mammals, but are described as causing persistent infection and lacking pathogenicity. The efficiency of in vitro replication systems for pegivirus is poor, thus limiting investigation into viral replication steps. Because of that, the pathogenesis, cellular tropism, route of transmission, biology, and epidemiology of pegiviruses remain largely uncovered. Here, we report a phylogenetically distinct goose pegivirus (GPgV) that should be classified as a new species. GPgV proliferated in cell culture in a species- and cell-type-specific manner. Animal experiments show GPgV lymphotropism and promote goose parvovirus clinical manifestations. This study provides the first cell culture model for pegivirus, opening new possibilities for studies of pegivirus molecular biology. More importantly, our findings stand in contrast to the lack of identified pathogenicity of previously reported pegiviruses, which sheds lights on the pathobiology of pegivirus.

Binding of Duck Tembusu Virus Nonstructural Protein 2A to Duck STING Disrupts Induction of Its Signal Transduction Cascade To Inhibit Beta Interferon Induction.

Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-ß and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-ß transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-ß inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.

cis-Acting Sequences and Secondary Structures in Untranslated Regions of Duck Tembusu Virus RNA Are Important for Cap-Independent Translation and Viral Proliferation.

Duck Tembusu virus (DTMUV) (genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. The positive-strand RNA genomes of flaviviruses are commonly translated in a cap-dependent manner. However, dengue and Zika viruses also exhibit cap-independent translation. In this study, we show that RNAs containing 5' and 3' untranslated regions (UTRs) of DTMUV, mosquito-borne Tembusu virus (TMUV), and Japanese encephalitis virus can be translated in a cap-independent manner in mammalian, avian, and mosquito cells. The ability of the 5' UTRs of flaviviruses to direct the translation of a second open reading frame in bicistronic RNAs was much less than that observed for internal ribosome entry site (IRES) encephalomyocarditis virus, indicating a lack of substantial IRES activity. Instead, cap-independent translation of DTMUV RNA was dependent on the presence of a 3' UTR, RNA secondary structures located in both UTRs, and specific RNA sequences. Mutations inhibiting cap-independent translation decreased DTMUV proliferation in vitro and delayed, but did not prevent, the death of infected duck embryos. Thus, the 5' and 3' UTRs of DTMUV enable the virus to use a cap- and IRES-independent RNA genome translation strategy that is important for its propagation and virulence.IMPORTANCE The genus Flavivirus includes major human pathogens, as well as animal-infecting viruses with zoonotic potential. In order to counteract the threats these viruses represent, it is important to understand their basic biology to develop universal attenuation strategies. Here, we demonstrate that five different flaviviruses use cap-independent translation, indicating that the phenomenon is probably common to all members of the genus. The mechanism used for flavivirus cap-independent translation was found to be different from that of IRES-mediated translation and dependent on both 5' and 3' UTRs that act in cis As cap-independent translation was also observed in mosquito cells, its role in flavivirus infection is unlikely to be limited to the evasion of consequences of the shutoff of host translation. We found that the inhibition of cap-independent translation results in decreased viral proliferation, indicating that the strategy could be applied to produce attenuated variants of flaviviruses as potential vaccine candidates.

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.

Binding of the Duck Tembusu Virus Protease to STING Is Mediated by NS2B and Is Crucial for STING Cleavage and for Impaired Induction of IFN-ß.

Duck Tembusu virus (DTMUV) is a newly emerged causative agent of avian disease. The protease-dependent immune evasion of flaviviruses has been reported; however, the molecular details of this process are unclear. In this study, we found that DTMUV nonstructural protein 2B-3, a NS2B3 protease, can inhibit IFN-ß production. DTMUV NS2B3 inhibited RIG-I-, MDA5-, MAVS-, and STING-directed IFN-ß transcription, but not TBK1- and IRF7-mediated induction of IFN-ß. Further analysis showed that DTMUV NS2B3 could cleave duck STING (duSTING); the cleavage was dependent on the protease activity of NS2B3. Moreover, the STING cleavage event occurred in a not-strictly-species-specific manner. The scissile bond of duSTING cleaved by NS2B3 was mapped between the R84 and G85 residues. The ability of NS2B3 to reduce duSTING cleavage-resistant mutant-mediated IFN-ß, and ISG production was significantly reduced, demonstrating that duSTING cleavage is essential for NS2B3-induced suppression of type I IFN responses. Remarkably, the binding of NS2B3 to duSTING, which is a prerequisite for cleavage, was found to depend on NS2B, but not NS3, the cofactor of the enzyme. Unexpectedly, we found that the region between aa residues 221-225 of duSTING, distal from the site of the scissile bond, was essential for the binding of NS2B3 to duSTING and/or the cleavage of duSTING by NS2B3. Thus, we identified the molecular mechanism by which DTMUV subverts the host innate immunity using its protease. More importantly, our study provides insight into NS2B3-mediated STING cleavage events in general.

Duck IFIT5 differentially regulates Tembusu virus replication and inhibits virus-triggered innate immune response.

Mammalian interferon-induced protein with tetratricopeptide repeats family proteins (IFITs) play important roles in host innate immune response to viruses. Recently, studies have shown that IFIT from poultry also plays a crucial part in antiviral function. This study first reports the regulation of duck Tembusu virus (DTMUV) replication by IFIT5 and the effect of duck IFIT5 (duIFIT5) on the innate immune response after DTMUV infection. Firstly, duIFIT5 was obviously increased in duck embryo fibroblast cells (DEFs) infected with DTMUV. Compared to the negative control, we found that in the duIFIT5-overexpressing group, the DTMUV titer at 24 h post infection (hpi) was significantly reduced, but the viral titer was strikingly increased at 48 hpi. Moreover, overexpression of duIFIT5 could significantly inhibit IFN-ß transcription and IFN-ß promoter activation at indicated time points after DTMUV infection. Further, in DTMUV-infected or poly(I:C)-stimulated DEFs, overexpression of duIFIT5 also significantly inhibited the activation of NF-κB and IRF7 promoters, as well as the activation of downstream IFN induced the interferon-stimulated response element (ISRE) promoter. Meanwhile, the transcription level of antiviral protein Mx, but not OASL, was obviously decreased at various time points. The opposite results were obtained by knockdown of duIFIT5 in DTMUV-infected or poly(I:C)-stimulated DEFs. Compared to the negative control, knockdown of duIFIT5 promoted DTMUV titer and DTMUV envelope (E) protein expression at 24 hpi, but DTMUV titer and E protein expression was markedly decreased at 48 hpi. Additionally, the promoters of IFN-ß, NF-κB, IRF7 and ISRE were significantly activated in the duIFIT5 knockdown group. Collectively, duIFIT5 differentially regulates DTMUV replication and inhibits virus-triggered innate immune response.

Heterologous prime-boost: an important candidate immunization strategy against Tembusu virus.

BACKGROUND: Tembusu virus (TMUV), a newly emerging pathogenic flavivirus, spreads rapidly between ducks, causing massive economic losses in the Chinese duck industry. Vaccination is the most effective method to prevent TMUV. Therefore, it is urgent to look for an effective vaccine strategy against TMUV. Heterologous prime-boost regimens priming with vaccines and boosting with recombinant adenovirus vaccines have been proven to be successful strategies for protecting against viruses in experimental animal models. METHODS: In this study, heterologous and homologous prime-boost strategies using an attenuated salmonella vaccine and a recombinant adenovirus vaccine expressing prM-E or the E gene of TMUV were evaluated to protect ducks against TMUV infection for the first time, including priming and boosting with the attenuated salmonella vaccine, priming and boosting with the recombinant adenovirus vaccine, and priming with the attenuated salmonella vaccine and boosting with the recombinant adenovirus vaccine. Humoral and cellular immune responses were detected and evaluated. We then challenged the ducks with TMUV at 12 days after boosting to assay for clinical symptoms, mortality, viral loads and histopathological lesions after these different strategies. RESULTS: Compared with the homologous prime-boost strategies, the heterologous prime-boost regimen produced higher levels of neutralizing antibodies and IgG antibodies against TMUV. Additionally, it could induce higher levels of IFN-γ than homologous prime-boost strategies in the later stage. Interestingly, the heterologous prime-boost strategy induced higher levels of IL-4 in the early stage, but the IL-4 levels gradually decreased and were even lower than those induced by the homologous prime-boost strategy in the later stage. Moreover, the heterologous prime-boost strategy could efficiently protect ducks, with low viral titres, no clinical symptoms and histopathological lesions in this experiment after challenge with TMUV, while slight clinical symptoms and histopathological lesions were observed with the homologous prime-boost strategies. CONCLUSIONS: Our results indicated that the heterologous prime-boost strategy induced higher levels of humoral and cellular immune responses and better protection against TMUV infection in ducks than the homologous prime-boost strategies, suggesting that the heterologous prime-boost strategy is an important candidate for the design of a novel vaccine strategy against TMUV.

Host shutoff activity of VHS and SOX-like proteins: role in viral survival and immune evasion.

BACKGROUND: Host shutoff refers to the widespread downregulation of host gene expression and has emerged as a key process that facilitates the reallocation of cellular resources for viral replication and evasion of host antiviral immune responses. MAIN BODY: The Herpesviridae family uses a number of proteins that are responsible for host shutoff by directly targeting messenger RNA (mRNA), including virion host shutoff (VHS) protein and the immediate-early regulatory protein ICP27 of herpes simplex virus types 1 (HSV-1) and the SOX (shutoff and exonuclease) protein and its homologs in Gammaherpesvirinae subfamilies, although these proteins are not homologous. In this review, we highlight evidence that host shutoff is promoted by the VHS, ICP27 and SOX-like proteins and that they also contribute to immune evasion. CONCLUSIONS: Further studies regarding the host shutoff proteins will not only contribute to provide new insights into the viral replication, expression and host immune evasion process, but also provide new molecular targets for the development of antiviral drugs and therapies.

The role of host eIF2α in viral infection.

BACKGROUND: eIF2α is a regulatory node that controls protein synthesis initiation by its phosphorylation or dephosphorylation. General control nonderepressible-2 (GCN2), protein kinase R-like endoplasmic reticulum kinase (PERK), double-stranded RNA (dsRNA)-dependent protein kinase (PKR) and heme-regulated inhibitor (HRI) are four kinases that regulate eIF2α phosphorylation. MAIN BODY: In the viral infection process, dsRNA or viral proteins produced by viral proliferation activate different eIF2α kinases, resulting in eIF2α phosphorylation, which hinders ternary tRNAMet-GTP-eIF2 complex formation and inhibits host or viral protein synthesis. The stalled messenger ribonucleoprotein (mRNP) complex aggregates under viral infection stress to form stress granules (SGs), which encapsulate viral RNA and transcription- and translation-related proteins, thereby limiting virus proliferation. However, many viruses have evolved a corresponding escape mechanism to synthesize their own proteins in the event of host protein synthesis shutdown and SG formation caused by eIF2α phosphorylation, and viruses can block the cell replication cycle through the PERK-eIF2α pathway, providing a favorable environment for their own replication. Subsequently, viruses can induce host cell autophagy or apoptosis through the eIF2α-ATF4-CHOP pathway. CONCLUSIONS: This review summarizes the role of eIF2α in viral infection to provide a reference for studying the interactions between viruses and hosts.

Duck enteritis virus pUL47, as a late structural protein localized in the nucleus, mainly depends on residues 40 to 50 and 768 to 777 and inhibits IFN-ß signalling by interacting with STAT1.

Duck enteritis virus (DEV) is a member of the Alphaherpesvirinae subfamily. The characteristics of some DEV genes have been reported. However, information regarding the DEV UL47 gene is limited. In this study, we identified the DEV UL47 gene encoding a late structural protein located in the nucleus of infected cells. We further found that two domains of DEV pUL47, amino acids (aa) 40 to 50 and 768 to 777, could function as nuclear localization sequence (NLS) to guide the nuclear localization of pUL47 and nuclear translocation of heterologous proteins, including enhanced green fluorescent protein (EGFP) and beta-galactosidase (ß-Gal). Moreover, pUL47 significantly inhibited polyriboinosinic:polyribocytidylic acid [poly(I:C)]-induced interferon beta (IFN-ß) production and downregulated interferon-stimulated gene (ISG) expression, such as Mx and oligoadenylate synthetase-like (OASL), by interacting with signal transducer and activator of transcription-1 (STAT1).

Duck enteritis virus UL21 is a late gene encoding a protein that interacts with pUL16.

BACKGROUND: pUL21 is a conserved protein of Alphaherpesvirinae that performs multiple important functions. The C-terminus of pUL21 in other members of this subfamily has RNA-binding ability; this domain contributes to pseudorabies virus (PRV) retrograde axonal transport in vitro and in vivo and participates in newly replicated viral DNA packaging and intracellular virus transport. However, knowledge regarding duck enteritis virus (DEV) pUL21 is limited. RESULTS: We verified that DEV UL21 is a γ2 gene that encodes a structural protein. Moreover, we observed that pUL21 localized to the nucleus and cytoplasm. DEV pUL21 interacted with pUL16 and formed a complex in transfected human embryonic kidney (HEK) 293 T cells and DEV-infected duck embryo fibroblasts (DEFs). These results were further confirmed by CO-IP assays. CONCLUSIONS: The DEV UL21 gene is a late gene, and pUL21 localizes to the nucleus and cytoplasm. DEV UL21 is a virion component. In addition, pUL21 can interact with pUL16. These findings provide insight into the characteristics of UL21 and the interaction between pUL21 and its binding partner pUL16. Our study enhances the understanding of DEV pUL21.

New Perspectives on Galleria mellonella Larvae as a Host Model Using Riemerella anatipestifer as a Proof of Concept.

Galleria mellonella larvae have been used as a host model to study interactions between pathogens and hosts for several years. However, whether the model is useful to interrogate Riemerella anatipestifer infection biology remained unknown. This study aimed to exploit the potential of G. mellonella larvae and reveal their limitations as a host model for R. anatipestifer infection. G. mellonella larvae were shown to be effective for virulence evaluations of different R. anatipestifer strains. Furthermore, the virulent strain R. anatipestifer CH-1 had a stronger ability to proliferate than the attenuated strain R. anatipestifer ATCC 11845 in both G. mellonella larvae and ducklings. Unconventionally it was shown that G. mellonella larvae cannot be used to evaluate the efficacy of antimicrobials and their combinations. Additionally, it was shown that certain virulence factors, such as OmpA (B739_0861), B739_1208, B739_1343, and Wza (B739_1124), were specific only for ducklings, suggesting that G. mellonella larvae must be cautiously used to identify virulence factors of R. anatipestifer Evaluation of heme uptake-related virulence genes, such as tonB1 and tonB2, required preincubating the strains with hemoglobin before infection of G. mellonella larvae since R. anatipestifer cannot obtain a heme source from G. mellonella larvae. In conclusion, this study revealed the applicability and limitations of G. mellonella as a model with which to study the pathogen-host interaction, particularly in the context of R. anatipestifer infection.

Prevalence of fluoroquinolone resistance and mutations in the gyrA, parC and parE genes of Riemerella anatipestifer isolated from ducks in China.

BACKGROUND: Riemerella anatipestifer is one of the most serious infectious disease-causing pathogens in the duck industry. Drug administration is an important method for prevention and treatment of infection in duck production, leading to widespread drug resistance in R. anatipestifer. METHODS: For a total of 162 isolates of R. anatipestifer, the MICs were determined for a quinolone antimicrobial agent, namely, nalidixic acid, and three fluoroquinolones, namely, ciprofloxacin, enrofloxacin and ofloxacin. The gyrA, parC, and parE gene fragments were amplified by PCR to identify the mutation sites in these strains. Site-directed mutants with mutations that were detected at a high frequency in vivo were constructed (hereafter referred to as site-directed in vivo mutants), and the MICs of these four drugs for these strains were determined. RESULTS: In total, 100, 97.8, 99.3 and 97.8% of the 137 R. anatipestifer strains isolated between 2013 and 2018 showed resistance to nalidixic acid, ciprofloxacin, enrofloxacin, and ofloxacin, respectively. The high-frequency mutation sites were detected in a total of 162 R. anatipestifer strains, such as Ser83Ile and Ser83Arg, which are two types of substitution mutations of amino acid 83 in GyrA; Val799Ala and Ile811Val in ParC; and Val357Ile, His358Tyr, and Arg541Lys in ParE. MIC analysis results for the site-directed in vivo mutants showed that the strains with only the Ser83Ile mutation in GyrA exhibited an 8-16-fold increase in MIC values, and all mutants showed resistance to ampicillin and ceftiofur. CONCLUSIONS: The resistance of R. anatipestifer to quinolone agents is a serious problem. Amino acid 83 in GyrA is the major target mutation site for the fluoroquinolone resistance mechanism of R. anatipestifer.

Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells.

BACKGROUND: Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. METHODS: In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. RESULTS: We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. CONCLUSIONS: Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV.

The 164 K, 165 K, and 167 K residues of VP1 are vital for goose parvovirus proliferation in GEFs based on PCR-based reverse genetics system.

BACKGROUND: Goose parvovirus (GPV) is the etiological agent of Derzsy's disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. METHODS: In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17-22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. RESULTS: The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. CONCLUSIONS: The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.

Downregulation of microRNA-30a-5p contributes to the replication of duck enteritis virus by regulating Beclin-1-mediated autophagy.

BACKGROUND: MicroRNAs (miRNAs) is increasingly recognized as an important element in regulating virus-host interactions. Our previous results showed that cellular miR-30a-5p was significantly downregulated after duck enteritis virus (DEV) infection cell. However, whehter or not the miR-30a-5p is involved in DEV infection has not been known. METHODS: Quantitative reverse-transcription PCR (qRT-PCR) was used to measure the expression levels of miRNAs(miR-30a-5p) and Beclin-1 mRNA. The miR-30a-5p - Beclin-1 target interactions were determined by Dual luciferase reporter assay (DLRA). Western blotting was utilized to analyze Beclin-1-mediated duck embryo fibroblast (DEF) cells autophagy activity. DEV titers were estimated by the median tissue culture infective dose (TCID50). RESULTS: The miR-30a-5p was significantly downregulated and the Beclin-1 mRNA was significantly upregulated in DEV-infected DEF cells. DLRA confirmed that miR-30a-5p directly targeted the 3'- UTR of the Beclin-1 gene. Overexpression of miR-30a-5p significantly reduced the expression level of Beclin-1protein (p < 0.05), leading to the decrease of Beclin-1-mediated autophagy activity, which ultimately suppressed DEV replication (P < 0.05). Whereas transfection of miR-30a-5p inhibitor increased Beclin-1-mediated autophagy and triggered DEV replication during the whole process of DEV infection (P < 0.01). CONCLUSIONS: This study shows that miR-30a-5p can inhibit DEV replication through reducing autophagy by targeting Beclin-1. These findings suggest a new insight into virus-host interaction during DEV infection and provide a potential new antiviral therapeutic strategy against DEV infection.

Class 1 integrons as predominant carriers in Escherichia coli isolates from waterfowls in Hainan, China.

This study was conducted to determine the prevalence of integrons and associated gene cassettes in Escherichia coli (E. coli) isolates from waterfowls in Hainan, China. The antimicrobial resistance profile of the isolates was examined by using disc diffusion test. In addition, PCR, RFLP, plasmid replicon typing and DNA sequencing analyses were used for the characterization of integrase genes (class 1, 2 and 3) and associated gene cassettes. Approximatively, 90% of the isolates were positive for the integrase genes by PCR. Specifically, class 1 and class 2 integrons were found in 252 (81%) and 7 (2.3%) strains, respectively. While 21 (6.7%) isolates were positive for both class 1 and class 2 integrons. However, none of the isolate was positive for the class 3 integrons. In addition, 5 various cassette arrays, dfrA1-orfC, aadA2, aadA1, dfrA1-aadA1, and dfrA1-orfC- aadA1, were found within the variable regions (VRs) of class 1 integron isolates. While only single cassette array, dfrA1-sat2- aadA1, was identified within VRs of class 2 integron isolates. We identified incF plasmid as the most common plasmid type, which was detected in 81 of 243 VRs containing isolates. This study is the first report showing the baseline characteristics of integrons in E. coli isolates from waterfowls in Hainan, China. Our results provide evidence of the waterfowl birds as a reservoir of class 1 and class 2 integrons carrying antibiotic resistance gene cassettes. Therefore, strict preventive measures should be taken to avoid the spread of mobile genetic resistance elements in waterfowls in China.