Zhijing Xie

Klebsiella pneumoniae infection following H9N2 Influenza A virus (IAV) infection causes severe pneumonia. But the underlying pathogenic mechanisms of H9N2 IAV and K. pneumoniae co-infection are complex and need to be further explored. In this study, the lung transcriptomes of mice with H9N2 IAV and K. pneumoniae co-infection were characterized by transcriptomic profiling. As a result, GO enrichment analysis revealed that the differential genes were primarily involved in the activation of immune responses, cellular components of membranes and extracellular spaces, and defense responses against pathogen infections. According to KEGG enrichment, the differentially expressed genes (DEGs) were concentrated in TLR signaling pathways, RLR signaling pathways, TNF signaling pathways and NLRP3 signaling pathways. Furthermore, in vitro cell models were established to investigate the innate immune responses induced by H9N2 IAV and K. pneumoniae CPS co-stimulation. K. pneumoniae CPS stimulation influenced the cytokine profiles of mink lung epithelial cells infected with H9N2 IAV, worsened cell viability, and aggravated apoptosis, indirectly inhibiting H9N2 IAV replication. The findings demonstrated that K. pneumoniae superinfection modulated the innate immune responses induced by H9N2 IAV infection, contributing to its pathogenesis.

With the continuous development of duck farming and the increasing breeding density, the incidence of duck hepatitis A virus type 1 (DHAV-1) has been on the rise, seriously endangering the development of duck farming. To reduce the use of antibiotics in duck breeding, susceptibility risks and mortality, and avoid virulence recovery and immune failure risk, this study aims to develop a new type of mucosal immune probiotics and make full use of molecular biology techniques, on the level of genetic engineering, to modify Lactococcus lactis (L. lactis). In this study, a secretory recombinant L. lactis named MG1363-VP1 with an enhanced Green Fluorescent Protein (eGFP) and translation enhancer T7g10L was constructed, which could express the VP1-eGFP fusion protein of DHAV-1. The animal experiment in ducklings was performed to detect the immune response and protection effect of oral microecologics by recombinant L. lactis. The results showed that oral L. lactis MG1363-VP1 significantly induced the body’s humoral immune system and mucosal immune system to produce specific anti-VP1 IgG antibodies and mucosal secretory immunoglobulin A (sIgA) for DHAV-1 in ducklings, and cytokines including interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10), and interferon gamma (IFN-γ). The mortality rate was monitored simultaneously by the natural infestation in the process of production and breeding; notably, the ducklings vaccinated with L. lactis MG1363-VP1 were effectively protected against the nature infection of DHAV-1. The recombinant L. lactis MG1363-VP1 constructed in this study provides a new means of preventing and controlling DHAV-1 infection in the future.

Colistin has been used as a growth promotant in livestock feed for many years. In China, mcr-1-positive Escherichia coli strains have been isolated from humans, chickens, and pigs. To date, there are few reports about the prevalence and molecular characteristics of fecal E. coli bearing mcr-1 in the meat ducks. In this study, the prevalence of mcr-1 gene was investigated among 120 fecal E. coli strains isolated from healthy meat ducks in Shandong province of China between October 2017 and February 2018. A total of nine mcr-1-containing E. coli strains were identified and two were identified as extra-intestinal pathogenic E. coli (ExPEC) among them. The clonal relationship of the nine E. coli strains was determined by multilocus sequencing typing (MLST) and pulsed field gel electrophoresis (PFGE), and the results indicated that all mcr-1-carrying isolates were clonally unrelated. Two different genetic contexts of mcr-1 were identified among these isolates. Colistin-resistant phenotype of all the isolates was successfully transferred to the recipient strains by conjugation experiments and seven transconjugants carried a single plasmid. The mcr-1 was located on three replicon plasmids: IncI2 (n = 4), IncFII (n = 2) and IncN (n = 1). Complete sequence analysis of a representative plasmid pTA9 revealed that it was strikingly similar with plasmid pMCR1-IncI2 of E. coli, plasmid pHNSHP45 of E. coli, and plasmid pWF-5-19C of Cronobacter sakazakii, implying that pTA9-like plasmids may be epidemic plasmids that mediate the spread of mcr-1 among Enterobacteriaceae. These results highlight that healthy meat duck is a potential reservoir for multidrug resistant mcr-1-containing E. coli strains.

Duck hepatitis A virus (DHAV), the major pathogen of duck virus hepatitis (DVH), causes severe diseases that threaten the duck industry worldwide. The VP1 protein, a major structural protein of DHAV, is able to induce neutralizing antibody in ducks. The purpose of this study was to identify the antigenic mimotope of DHAV by phage display technology. A monoclonal antibody (mAb) 4E6 against DHAV-1 and DHAV-3 was prepared, and a phage library prepared with the PhD-12 Phage Display Peptide Library Kit was screened with the mAb. A novel peptide, ¹GLTWKLPPSM¹⁰ was identified with high affinity to the mAb and could specifically block mAb 4E6 from binding DHAV-1 and DHAV-3. Animal tests confirmed that the immunization of ducklings with the mimotope could inhibit the virus proliferation and protect the ducklings from DVH. In summary, the neutralizing conformational mimotope ¹GLTWKLPPSM¹⁰ might be a promising vaccine candidate for the prevention of DHAV infection.

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.

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.

DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay. A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 μg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 μg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings. Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest. We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.