The innate immune system provides a defense against invading pathogens by inducing various interferon (IFN)-stimulated genes (ISGs). We recently reported that tripartite motif protein 25 (TRIM25), an important ISG, was highly upregulated in duck embryo hepatocyte cells (DEFs) after infection with duck viral hepatitis A virus type 1 (DHAV-1). However, the mechanism of upregulation of TRIM25 remains unknown. Here we reported that interleukin-22 (IL-22), whose expression was highly facilitated in DEFs and various organs of 1-day-old ducklings after DHAV-1 infection, highly enhanced the IFN-λ-induced production of TRIM25. The treatment with IL-22 neutralizing antibody or the overexpression of IL-22 highly suppressed or facilitated TRIM25 expression, respectively. The phosphorylation of signal transducer and activator of transcription 3 (STAT3) was crucial for the process of IL-22 enhancing IFN-λ-induced TRIM25 production, which was suppressed by WP1066, a novel inhibitor of STAT3 phosphorylation. The overexpression of TRIM25 in DEFs resulted in a high production of IFNs and reduced DHAV-1 replication, whereas the attenuated expression of IFNs and facilitated replication of DHAV-1 were observed in the RNAi group, implying that TRIM25 defended the organism against DHAV-1 propagation by inducing the production of IFNs. In summary, we reported that IL-22 activated the phosphorylation of STAT3 to enhance the IFN-λ-mediated TRIM25 expression and provide a defense against DHAV-1 by inducing IFN production.

The interaction between viral components and cellular proteins plays a crucial role in viral replication. In a previous study, we showed that the 3′—untranslated region (3′—UTR) is an essential element for the replication of duck hepatitis A virus type 1 (DHAV-1). However, the underlying mechanism is still unclear. To gain a deeper understanding of this mechanism, we used an RNA pull-down and a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay to identify new host factors that interact with the 3′—UTR. We selected interleukin-2 enhancer binding factor 2 (ILF2) for further analysis. We showed that ILF2 interacts specifically with both the 3′—UTR and the 3D polymerase (3Dpol) of DHAV-1 through in vitro RNA pull-down and co-immunoprecipitation assays, respectively. We showed that ILF2 negatively regulates viral replication in duck embryo fibroblasts (DEFs), and that its overexpression in DEFs markedly suppresses DHAV-1 replication. Conversely, ILF2 silencing resulted in a significant increase in viral replication. In addition, the RNA-dependent RNA polymerase (RdRP) activity of 3Dpol facilitated viral replication by enhancing viral RNA translation efficiency, whereas ILF2 disrupted the role of RdRP in viral RNA translation efficiency to suppress DHAV-1 replication. At last, DHAV-1 replication markedly suppressed the expression of ILF2 in DEFs, duck embryo hepatocytes, and different tissues of 1 day-old ducklings. A negative correlation was observed between ILF2 expression and the viral load in primary cells and different organs of young ducklings, suggesting that ILF2 may affect the viral load both in vitro and in vivo.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a bunyavirus that causes SFTS, with a case fatality rate of up to 30 %. The innate immune system plays a crucial role in the defense against SFTSV; however, the impact of viral propagation of STFSV on the innate immune system remains unclear. Although proteomics analysis revealed that the expression of the downregulator of transcription 1 (DR1) increased after SFTSV infection, the specific change trend and the functional role of DR1 during viral infection remain unelucidated. In this study, we demonstrate that DR1 was highly expressed in response to SFTSV infection in HEK 293T cells using qRT-PCR and Western blot analysis. Furthermore, viral replication significantly increased the expression of various TLRs, especially TLR9. Our data indicated that DR1 positively regulated the expression of TLRs in HEK 293T cells, DR1 overexpression highly increased the expression of numerous TLRs, whereas RNAi-mediated DR1 silencing decreased TLR expression. Additionally, the myeloid differentiation primary response gene 88 (MyD88)-dependent or TIR-domain-containing adaptor inducing interferon-β (TRIF)-dependent signaling pathways were highly up- and downregulated by the overexpression and silencing of DR1, respectively. Finally, we report that DR1 stimulates the expression of TLR7, TLR8, and TLR9, thereby upregulating the TRIF-dependent and MyD88-dependent signaling pathways during the SFTSV infection, attenuating viral replication, and enhancing the production of type I interferon and various inflammatory factors, including IL-1β, IL-6, and IL-8. These results imply that DR1 defends against SFTSV replication by inducing the expression of TLR7, TLR8, and TLR9. Collectively, our findings revealed a novel role and mechanism of DR1 in mediating antiviral responses and innate immunity.

Mercury chloride can cause severe liver injury, which involves multiple mechanisms. Ferroptosis plays an important role in regulating the development and progression of liver pathology. Oleanolic acid (OA), a tri-terpenoid compound widely exists in fruits, has liver protective properties. In this study, we investigated the role of ferroptosis in mercury chloride-induced liver injury and the intervention effect of OA, and clarified the po-tential mechanism. We found that mercury chloride-induced oxidative stress in liver tissues and cells, leading to lipid peroxidation and iron overload, thereby reducing the expression levels of GPX4 and SLC7A11, and increasing the expression level of TRF1, OA pretreatment improved the changes of GPX4, SLC7A11 and TRF1 induced by mercury chloride, which were related to its inhibition of oxidative stress. Furthermore, We pretreated cells with OA, VC, and Fer-1, respectively and found that VC pretreatment reduced oxidative stress and signif-icantly reversed the gene and protein expressions of GPX4, SLC7A11, and TRF1 in mercury chloride-exposed cells (P < 0.05, vs. HgCl2 group), however, the protein expression level of GPX4 in OA pre-treatment group was lower than that in VC pre-treatment group (P < 0.05). Fer-1 pretreatment decreased the level of iron ions in cells, increased the gene and protein expression levels of GPX4 and SLC7A11, and decreased the gene and protein expression levels of TRF1 (P < 0.05, vs. HgCl2 group), however, the protein expression levels of GPX4 and SLC7A11 in OA pre-treatment group were lower than those in Fer-1 pre-treatment group (P < 0.05). Moreover, vivo experiments also demonstrated that pre-treatment with OA, VC, and Fer-1 reversed the changes in gene expression levels of Nrf2 and SOD1, and protein expression of GPX4 induced by mercury chloride (P < 0.05, vs. HgCl2 group), meanwhile, the difference was not statistically significant among OA, VC, and Fer-1 pretreatment. The improvement effect of OA pretreatment on the change in TFR1 protein expression caused by mercury chloride was similar to that of Fer-1 and VC, however, the intervention effect of OA on SLC7A11 protein expression was not as good as Fer-1 and VC pre-treatment. To sum up, all these results suggest that ferroptosis is involved in mercury chloride-induced liver injury, OA pretreatment alleviated mercury chloride-induced fer-roptosis by inhibiting ROS production and iron ion overload, and then alleviate the liver injury.

Jingmen tick virus (JMTV) is a novel tick‐borne virus first identified from Jingmen city, Hubei Province of China in 2010. It has been proved that JMTV can cause human diseases and is widely distributed both inside and outside of China. However, the survival mode and transmission characteristics of JMTV still need further research, particularly in terms of transovarial transmission. In this study, an investigation was conducted to explore the presence of JMTV from engorged female ticks to their offspring. All engorged female adult ticks were collected from domestic cattle and allowed to lay eggs in appropriate humidity and temperature conditions. Maternal ticks, eggs and larvae were screened for JMTV RNA through real‐time polymerase chain reaction (RT‐PCR) and nested PCR methods. The results revealed the positive rate of 10.53% (10/95) in engorged ticks, 9.09% (2/22) in eggs and 8% (4/50) in larvae pools, respectively. Phylogenetic analysis confirmed that sequences from eggs and larvae had closer relationship with those isolates from maternal engorged ticks with more than 99.7% homology and JMTV manifested with evolutional conservatism. Our study has identified for the first time that JMTV could be transmitted from mother generation to offspring of Haemaphysalis Longicornis . Nonetheless, the efficiency of transovarial transmission in JMTV and the significance of ticks as amplification hosts still need to be further illustrated.

The placenta-specific 8 (PLAC8) gene, also known as ONZIN or C15, codes for a cysteine-rich peptide originally identified in mouse placental tissue and subsequently identified in a variety of epithelial tissues and immune cells. PLAC8 is also expressed in birds, such as ducks, where its functional roles remain unknown. Here, we aimed to determine the mRNA and protein expression profiles and the functional role of duck PLAC8 during the infection of duck hepatitis A virus type 1 (DHAV-1). We found that the duck PLAC8 is also a cysteine-rich polypeptide composed of 114 amino acid residues, with no signal peptide. Duck PLAC8 is highly expressed in the immune organs of young cherry valley ducks, including the thymus, bursa fabricius, and spleen. However, it has negligible expression level in liver, brain, kidney, and heart. Additionally, PLAC8 expression was considerably induced after DHAV-1 infection both in vitro and in vivo, especially in the immune organs of ducklings. This tissue expression distribution and induction upon infection suggest that PLAC8 might play a critical role in innate immunity. Our data showed that PLAC8 significantly suppressed the expression of Toll-like receptor 7 (TLR7), leading to decreased expression of downstream signaling molecules including myeloid differentiation primary response gene 88 (MyD88) and nuclear factor kappa-B (NF-κB). This ultimately resulted in low levels of type I interferon and interleukin 6 (IL-6). Additionally, PLAC8 positively regulated DHAV-1 replication levels. RNAi against PLAC8 in duck embryo fibroblasts considerably inhibited DHAV-1 propagation, while PLAC8 overexpression significantly facilitated DHAV-1 replication.

Summary Bacterial vectors can be engineered to generate microscopic living therapeutics to produce and deliver anticancer agents. Escherichia coli Nissle 1917 (Nissle 1917) is a promising candidate with probiotic properties. Here, we used Nissle 1917 to develop a metabolic strategy to produce 5‐aminolevulinic acid (5‐ALA) from glucose as 5‐ALA plays an important role in the photodynamic therapy of cancers. The coexpression of hemAM and hemL using a low copy‐number plasmid led to remarkable accumulation of 5‐ALA. The downstream pathway of 5‐ALA biosynthesis was inhibited by levulinic acid (LA). Small‐scale cultures of engineered Nissle 1917 produced 300 mg l−1 of 5‐ALA. Recombinant Nissle 1917 was applied to deliver 5‐ALA to colorectal cancer cells, in which it induced the accumulation of antineoplastic protoporphyrin X (PpIX) and specific cytotoxicity towards colorectal cancer cells irradiated with a 630 nm laser. Moreover, this novel combination therapy proved effective in a mouse xenograft model and was not cytotoxic to normal tissues. These findings suggest that Nissle 1917 will serve as a potential carrier to effectively deliver 5‐ALA for cancer therapy.

Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 μg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.

Duck astrovirus type 1 (DAstV-1) infection constitutes a cause of viral hepatitis in ducklings and little is known about the B-cell epitope of DAstV-1. In this study, a monoclonal antibody (mAb) 3D2 against open reading frame 2 (ORF2) protein of DAstV-1 was used to identify the possible epitope in the four serotypes of DAstV. The mAb 3D2 showed no neutralization activity to DAstV-1, and reacted with the conserved linear B-cell epitopes of 454STTESA459 in DAstV-1 ORF2 protein. Sequence analysis, dot blot assay, and cross-reactivity test indicated that the epitope peptide was highly conserved in DAstV-1 sequence and mAb 3D2 had no cross-reactivity with other DAstV serotypes. To the best of our knowledge, this is the first report about identification of the specific conserved linear B-cell epitope of DAstV-1, which will facilitate the serologic diagnosis of DAstV-1 infection.

As a disease characterized by severe liver necrosis and hemorrhage, duck viral hepatitis (DVH) is mainly caused by duck hepatitis A virus (DHAV). The positive-strand RNA genome of DHAV type 1 (DHAV-1) contains an internal ribosome entry site (IRES) element within the 5' untranslated region (UTR), structured sequence elements within the 3' UTR, and a poly(A) tail at the 3' terminus. In this study, we first examined that insulin-like growth factor-2 mRNA-binding protein-1 (IGF2BP1) specifically interacted with the DHAV-1 3' UTR by RNA pull-down assay. The interaction between IGF2BP1 and DHAV-1 3' UTR strongly enhanced IRES-mediated translation efficiency but failed to regulate DHAV-1 replication in a duck embryo epithelial (DEE) cell line. The viral propagation of DHAV-1 strongly enhanced IGF2BP1 expression level, and viral protein accumulation was identified as the key point to this increment. Collectively, our data demonstrated the positive role of IGF2BP1 in DHAV-1 viral proteins translation and provided data support for the replication mechanism of DHAV-1.

The nuclear localization signals (NLS) were usually composed of basic residues (K and R) and played an important role in delivery of genomes and structural protein into nucleus. In this research, we identified that 3Dpol/3CD entered into nucleus during viral propagation of duck hepatitis A virus type 1 (DHAV-1). To investigate the reason that 3Dpol/3CD entered into nucleus, the amino acid sequence of 3CD was analyzed through NLS Mapper program. The basic region 17PRKTAYMRS25 was subsequently proved to be a functional NLS to guide 3Dpol/3CD into nucleus. 18R, 19K and 24R were found essential for maintaining the nuclear targeting activity, and exchange between 24R and 24K had no impact on cellular localization of 3Dpol. Since the entry of 3Dpol/3CD into nucleus was essential for shutoff of host cell transcription and maintaining the viral propagation of picornavirus numbers, our study provided new insights into the mechanism of DHAV-1 propagation.

Duck hepatitis A virus (DHAV) is the major pathogen of duck viral hepatitis, which has caused great economic losses to duck breeding industry. As an effective delivery tool for protein antigens, Lactococcus lactis (L. lactis) has been successfully used to stimulate mucosal and systemic immune response. In this study, a recombinant L. lactis named NZ3900-VP1 was constructed, which could express VP1 protein of DHAV type 3 (DHAV-3) by using a nisin-controlled expression (NICE) system. The animal experiment in both mice and ducklings were performed to detect the immune response and protection effect of oral vaccination by the recombinant L. lactis. The results showed that oral vaccination with L. lactis NZ3900-VP1 significantly induced specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) of DHAV-3 in mice and ducklings, and cytokines including interleukin-2 (IL-2), interferon gamma (IFN-γ), interleukin-10 (IL-10) and interleukin-4 (IL-4). Notably, the ducklings vaccinated with L. lactis NZ3900-VP1 were effectively protected when facing natural infestation of DHAV-3, which indicated that the recombinant L. lactis could serve as an effective vaccine to prevent DHAV-3 infection in ducklings.

为了解猪嵴病毒(PKV)在山东省内的流行情况,本研究根据PKV的3D基因保守区域设计一对引物,对2014年冬～2016年冬来自山东省350份病料样品进行RT-PCR检测和统计分析.结果显示:在350份病料样品中,PKV阳性率为56％(196/350),阳性猪场占80％(24/30);非腹泻猪个体阳性率(65.38％,153/234)明显高于腹泻猪个体阳性率(37.07％,43/116);育肥猪的PKV阳性率(97.50％)远高于哺乳期仔猪(45.40％)、断奶期仔猪(52.75％)以及成年猪(66.67％);夏秋季节采集的样品PKV检出率(71.94％)明显高于冬春季节(45.50％).统计PKV与PEDV、TGEV混合感染结果显示,虽然PKV在非腹泻猪群中单独感染率(61.11％,143/234)远高于腹泻猪群(18.97％,22/116),但非腹泻猪群PKV与PEDV和TGEV的混合感染率却明显低于腹泻猪群.对5份PKV阳性样品的31D基因测序分祈显示,5份阳性样品该基因核苷酸同源性为90.8 ％～99.7％,与GenBank中的参考株之间的核苷酸同源性为88.8 ％～94.1％.上述研究结果表明在山东省范围内PKV感染与猪腹泻之间无必然关系,PKV的3D基因进化与病毒株的分离年代和地域无明显相关性.

The duck hepatitis A virus type 1 (DHAV-1) is a member of Picornaviridae family, the genome of the virus contains a 5′ untranslated region (5′ UTR), a large open reading frame that encodes a polyprotein precursor and a 3′ UTR followed by a poly(A) tail. The translation initiation of virus proteins depends on the internal ribosome-entry site (IRES) element within the 5′ UTR. So far, little information is known about the role of the 3′ UTR and poly(A) tail during the virus proliferation. In this study, the function of the 3′ UTR and poly(A) tail of DHAV-1 in viral replication and IRES-mediated translation was investigated. The results showed that both 3′ UTR and poly(A) tail are important for maintaining viral genome RNA stability and viral genome replication. During DHAV-1 proliferation, at least 20 adenines were required for the optimal genome replication and the virus replication could be severely impaired when the poly (A) tail was curtailed to 10 adenines. In addition to facilitating viral genome replication, the presence of 3′ UTR and poly(A) tail significantly enhance IRES-mediated translation efficiency. Furthermore, 3′ UTR or poly(A) tail could function as an individual element to enhance the DHAV-1 IRES-mediated translation, during which process, the 3′ UTR exerts a greater initiation efficiency than the poly(A)25 tail.

2015年以来山东、江苏等地的樱桃谷肉鸭暴发了一种由新型鹅细小病毒(NGPV)引起的短喙-侏儒综合征(BADS)的疾病,造成了严重的经济损失.为探究该病毒在鸭体内分布规律,本研究根据GenBank已登录的NGPV的VP3基因设计了3条引物,建立了检测NGPV的半套式PCR方法.该方法对其它常见鸭病原微生物无特异扩增,特异性好;最低可检出100拷贝/μL的病毒核酸,灵敏度高.采用建立的半套式PCR方法对山东、江苏两地8个樱桃谷肉鸭鸭群120只病死鸭的心脏、肝脏、脾脏、肺脏、肾脏、胸腺、法氏囊、胰腺、脑和胆汁进行检测,结果显示患病鸭群的群体检出率为100％,个体检出率为80.8％(97/120);10种组织脏器中均检出NGPV,其中心脏检出率最高为93.8％,脑组织检出率最低为9.3％.本研究首次对NGPV在鸭体内10个脏器组织的分布进行统计分析,证明了NGPV对樱桃谷鸭具有广泛的组织嗜性.

近年来,我国大陆地区暴发了一种由新型鹅细小病毒(NGPV)感染引起的鸭“短喙侏儒综合征”的疫病.为了建立一种特异性好、灵敏度高适合临床大批量样本检测的方法,本试验将鸭源NGPV的VP3保守区域1段长390 bp的序列克隆制成核酸探针.特异性试验结果表明,制备的该核酸探针特异性高,只与鹅细小病毒(GPV)反应;灵敏性试验结果表明,针对NGPV最低检出量约为6 pg.应用该核酸探针对来自山东、江苏、安徽3省的87只疑患“短喙长舌-侏儒综合征”的樱桃谷肉鸭的870份组织脏器进行检测,结果个体阳性率为89.7％(78/87),检测的10种组织脏器中均有NGPV检出,说明该病毒对樱桃谷鸭具有广泛的组织嗜性.同时,利用PCR方法对采集的样品进行检测,比较发现与核酸探针法的阳性个体检出率及阳性个体检出符合率为100％.以上结果表明,制备的NGPV核酸探针特异性强,灵敏性高,适合不同来源的NGPV临床批量诊断和流行病学调查.

Generally, duck hepatitis A virus type 1 (DHAV-1) only infects young ducklings. Since December 2016, severe outbreaks of duck viral infection with egg drop, feed consumption decline, and ovary-oviduct disease have occurred in some laying duck flocks in Shandong Province of China. DHAV-1 isolated from the affected ducks was confirmed as the causative pathogen of the egg drop. Compared with other DHAV-1 strains, the novel isolate has three special amino acid mutation points in the most variable regions at the C-terminus of VP1. The experimental infection in laying ducks indicated that successful immunization with DHAV-1 vaccine could protect laying duck from infection. To the best of our knowledge, this is the first reported incidence of a severe duck disease outbreak involving egg drop syndrome caused by DHAV-1.