Xinxin Zhao

Type B Chloramphenicol Acetyltransferases Are Responsible for Chloramphenicol Resistance in Riemerella anatipestifer, China

Riemerella anatipestifer causes serositis and septicaemia in domestic ducks, geese, and turkeys. Traditionally, the antibiotics were used to treat this disease. Currently, our understanding of R. anatipestifer susceptibility to chloramphenicol and the underlying resistance mechanism is limited. In this study, the cat gene was identified in 69/192 (36%) R. anatipestifer isolated from different regions in China, including R. anatipestifer CH-2 that has been sequenced in previous study. Sequence analysis suggested that there are two copies of cat gene in this strain. Only both two copies of the cat mutant strain showed a significant decrease in resistance to chloramphenicol, exhibiting 4 μg/ml in the minimum inhibitory concentration for this antibiotic, but not for the single cat gene deletion strains. Functional analysis of the cat gene via expression in Escherichia coli BL21 (DE3) cells and in vitro site-directed mutagenesis indicated that His79 is the main catalytic residue of CAT in R. anatipestifer. These results suggested that chloramphenicol resistance of R. anatipestifer CH-2 is mediated by the cat genes. Finally, homology analysis of types A and B CATs indicate that R. anatipestifer comprises type B3 CATs.

Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins

Among the few non-structural proteins encoded by the picornaviral genome, the 2A protein is particularly special, irrespective of structure or function. During the evolution of the Picornaviridae family, the 2A protein has been highly non-conserved. We believe that the 2A protein in this family can be classified into at least five distinct types according to previous studies. These five types are (A) chymotrypsin-like 2A, (B) Parechovirus-like 2A, (C) hepatitis-A-virus-like 2A, (D) Aphthovirus-like 2A, and (E) 2A sequence of the genus Cardiovirus. We carried out a phylogenetic analysis and found that there was almost no homology between each type. Subsequently, we aligned the sequences within each type and found that the functional motifs in each type are highly conserved. These different motifs perform different functions. Therefore, in this review, we introduce the structures and functions of these five types of 2As separately. Based on the structures and functions, we provide suggestions to combat picornaviruses. The complexity and diversity of the 2A protein has caused great difficulties in functional and antiviral research. In this review, researchers can find useful information on the 2A protein and thus conduct improved antiviral research.

Contribution of RaeB, a Putative RND-Type Transporter to Aminoglycoside and Detergent Resistance in Riemerella anatipestifer

Riemerella anatipestifer is an important pathogenic bacterium that infects ducks. It exhibits resistance to multiple classes of antibiotics. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens and they are poorly understood in R. anatipestifer. In this study, a gene encoding the B739_0873 protein in R. anatipestifer CH-1, which belongs to the resistance-nodulation-cell division (RND) efflux pump family, was identified. With respect to the substrate specificity of B739_0873, the antibiotic susceptibility testing showed that the B739_0873 knockout strain was more sensitive to aminoglycosides and detergents than the wild-type strain. The transcription of B739_0873 was up-regulated when R. anatipestifer CH-1 was exposed to sub-inhibitory levels of these substrates. From the gentamicin accumulation assay, we concluded that B739_0873 was coupled to the proton motive force to pump out gentamicin. Furthermore, site-directed mutagenesis demonstrated that Asp 400, Asp 401, Lys 929, Arg 959, and Thr 966 were the crucial function sites of B739_0873 in terms of its ability to extrude aminoglycosides and detergents. Finally, we provided evidence that B739_0873 is co-transcribed with B739_0872, and that both B739_0872 and B739_0873 are required for aminoglycoside and detergent resistance. In view of these results, we designate B739_0873 as RaeB (Riemerella anatipestifer efflux).

Two Novel Salmonella Bivalent Vaccines Confer Dual Protection against Two Salmonella Serovars in Mice

Non-typhoidal Salmonella includes thousands of serovars that are leading causes of foodborne diarrheal illness worldwide. In this study, we constructed three bivalent vaccines for preventing both Salmonella Typhimurium and Salmonella Newport infections by using the aspartate semialdehyde dehydrogenase (Asd)-based balanced-lethal vector-host system. The constructed Asd+ plasmid pCZ11 carrying a subset of the Salmonella Newport O-antigen gene cluster including the wzx-wbaR-wbaL-wbaQ-wzy-wbaW-wbaZ genes was introduced into three Salmonella Typhimurium mutants: SLT19 (Δasd) with a smooth LPS phenotype, SLT20 (Δasd ΔrfbN) with a rough LPS phenotype, and SLT22 (Δasd ΔrfbN ΔpagL::T araC PBAD rfbN) with a smooth LPS phenotype when grown with arabinose. Immunoblotting demonstrated that SLT19 harboring pCZ11 [termed SLT19 (pCZ11)] co-expressed the homologous and heterologous O-antigens; SLT20 (pCZ11) exclusively expressed the heterologous O-antigen; and when arabinose was available, SLT22 (pCZ11) expressed both types of O-antigens, while in the absence of arabinose, SLT22 (pCZ11) expressed only the heterologous O-antigen. Exclusive expression of the heterologous O-antigen in Salmonella Typhimurium decreased the swimming ability of the bacterium and its susceptibility to polymyxin B. Next, the crp gene was deleted from the three recombinant strains for attenuation purposes, generating the three bivalent vaccine strains SLT25 (pCZ11), SLT26 (pCZ11), and SLT27 (pCZ11), respectively. Groups of BALB/c mice (12 mice/group) were orally immunized with 109 CFU of each vaccine strain twice at an interval of 4 weeks. Compared with a mock immunization, immunization with all three vaccine strains induced significant serum IgG responses against both Salmonella Typhimurium and Salmonella Newport LPS. The bacterial loads in the mouse tissues were significantly lower in the three vaccine-strain-immunized groups than in the mock group after either Salmonella Typhimurium or Salmonella Newport lethal challenge. All of the mice in the three vaccine-immunized groups survived the lethal Salmonella Typhimurium challenge. In contrast, SLT26 (pCZ11) and SLT27 (pCZ11) conferred full protection against lethal Salmonella Newport challenge, but SLT25 (pCZ11) provided only 50% heterologous protection. Thus, we developed two novel Salmonella bivalent vaccines, SLT26 (pCZ11) and SLT27 (pCZ11), suggesting that the delivery of a heterologous O-antigen in attenuated Salmonella strains is a prospective approach for developing Salmonella vaccines with broad serovar coverage.

Duck stimulator of interferon genes plays an important role in host anti-duck plague virus infection through an IFN-dependent signalling pathway

Graphical abstract DuSTING plays an important role in IFN&bgr; and ISGs producing. Figure. No Caption available. HighlightsOverexpression of duSTING can stimulate the production of cytokines.DuSTING plays an important role in type I IFN signaling pathway.DuSTING inhibits the replication of duck plague virus was initially explored. Abstract The human stimulator of interferon gene (STING) is an important molecule in innate immunity that stimulates type I interferon (IFN) production. However, the role of duck STING (duSTING) in innate immunity has yet to be explained. In this study, the full length of the duSTING cDNA sequence (1149 bp), which encodes 382 amino acid (aa) residues, was reported and showed the highest sequence similarity with chicken STINGs. The phylogenetic analysis based on STING aa showed that duSTING was grouped onto the birds clade. According to the tissue distribution spectrum analysis, duSTING was highly present in the bursa of Fabricius, glandular stomach, liver, pancreas, and small intestine of ducklings, as well as in the blood and pancreas of the adult duck. DuSTING mainly colocalized with the endoplasmic reticulum (ER) and mitochondria in transfected Baby Hamster Syrian Kidney (BHK21) and duck embryo fibroblasts (DEF) cells by an indirect immunofluorescence assay. The transfection of the DEFs with duSTING activated NF‐&kgr;B, which induced the transcription of IFN‐&bgr;, and the activated IFN induced the interferon‐stimulated response element (ISRE). Furthermore, the overexpression of duSTING significantly upregulated the mRNA level of duck IFN‐&bgr; and IFN‐stimulated genes (ISGs), such as duMx and duOASL and inhibited the replication of the double‐stranded DNA duck plague virus (DPV) in vitro. In addition, the knockdown of endogenous duSTING by shRNA significantly reduced the poly (I:C) (pIC), poly (dA:dT), and Tembusu virus (TMUV), induced IFN‐&bgr; production and significantly promoted DPV replication in vitro. In general, these data demonstrate that duSTING is vital for duck type I interferon induction and plays an important role in the host defence of DPV infection.

A novel resistance gene, lnu(H), conferring resistance to lincosamides in Riemerella anatipestifer CH-2

The Gram-negative bacterium Riemerella anatipestifer CH-2 is resistant to lincosamides, having a lincomycin (LCM) minimum inhibitory concentration (MIC) of 128 µg/mL. The G148_1775 gene of R. anatipestifer CH-2, designated lnu(H), encodes a 260-amino acid protein with ≤41% identity to other reported lincosamide nucleotidylyltransferases. Escherichia coli RosettaTM (DE3) containing the pBAD24-lnu(H) plasmid showed four- and two-fold increases in the MICs of LCM and clindamycin (CLI), respectively. A kinetic assay of the purified Lnu(H) enzyme for LCM and CLI showed that the protein could inactive lincosamides. Mass spectrometry analysis demonstrated that the Lnu(H) enzyme catalysed adenylylation of lincosamides. In addition, an lnu(H) gene deletion strain exhibited 512- and 32-fold decreases in LCM and CLI MICs, respectively. The wild-type level of lincosamide resistance could be restored by complementation with a shuttle plasmid carrying the lnu(H) gene. The transformant R. anatipestifer ATCC 11845 [lnu(H)] acquired by natural transformation also exhibited high-level lincosamide resistance. Moreover, among 175 R. anatipestifer field isolates, 56 (32.0%) were positive for the lnu(H) gene by PCR. In conclusion, Lnu(H) is a novel lincosamide nucleotidylyltransferase that inactivates LCM and CLI by nucleotidylylation, thus conferring high-level lincosamide resistance to R. anatipestifer CH-2.

The 3D protein of duck hepatitis A virus type 1 binds to a viral genomic 3′ UTR and shows RNA-dependent RNA polymerase activity

To explore the RNA-dependent RNA polymerase (RdRP) function of the 3D protein of duck hepatitis A virus type 1 (DHAV-1), the gene was cloned into the pET-32a(+) vector for prokaryotic expression. The 3′ untranslated region (3′ UTR) of DHAV-1 together with a T7 promoter was cloned into the pMD19-T vector for in vitro transcription of 3′ UTR RNA, which was further used as a template in RNA-dependent RNA polymerization. In this study, three methods were applied to analyze the RdRP function of the 3D protein: (1) ammonium molybdate spectrophotometry to detect pyrophosphate produced during polymerization; (2) quantitative reverse transcription PCR (RT-qPCR) to investigate the changes in RNA quantity during polymerization; and (3) electrophoresis mobility shift assay to examine the interaction between the 3D protein and 3′ UTR. The results showed the 3D protein was successfully expressed in bacteria culture supernatant in a soluble form, which could be purified by affinity chromatography. In 3D enzymatic activity assays, pyrophosphate and RNA were produced, the amounts of which increased based on approximative kinetics, and binding of the 3D protein to the 3′ UTR was observed. These results indicate that prokaryotically expressed soluble DHAV-13D protein can bind to a viral genomic 3′ UTR and exhibit RdRP activity.

Regulation of viral gene expression by duck enteritis virus UL54

Duck enteritis virus (DEV) UL54 is a homologue of human herpes simplex virus-1 (HSV-1) ICP27, which plays essential regulatory roles during infection. Our previous studies indicated that DEV UL54 is an immediate-early protein that can shuttle between the nucleus and the cytoplasm. In the present study, we found that UL54-deleted DEV (DEV-ΔUL54) exhibits growth kinetics, a plaque size and a viral DNA copy number that are significantly different from those of its parent wild-type virus (DEV-LoxP) and the revertant (DEV-ΔUL54 (Revertant)). Relative viral mRNA levels, reflecting gene expression, the transcription phase and the translation stage, are also significantly different between DEV-ΔUL54-infected cells and DEV-LoxP/DEV-ΔUL54 (Revertant)-infected cells. However, the localization pattern of UL30 mRNA is obviously changed in DEV-ΔUL54-infected cells. These findings suggest that DEV UL54 is important for virus growth and may regulate viral gene expression during transcription, mRNA export and translation.

Molecular characterization of the duck enteritis virus US10 protein

BackgroundThere is little information regarding the duck enteritis virus (DEV) US10 gene and its molecular characterization.MethodsDuck enteritis virus US10 was amplified and cloned into the recombinant vector pET32a(+). The recombinant US10 protein was expressed in Escherichia coli BL21 cells and used to immunize rabbits for the preparation of polyclonal antibodies. The harvested rabbit antiserum against DEV US10 was detected and analyzed by agar immunodiffusion. Using this antibody, western blotting and indirect immunofluorescence analysis were used to analyze the expression level and subcellular localization of US10 in infected cells at different time points. Quantitative reverse-transcription PCR (qRT-PCR) and pharmacological inhibition tests were used to ascertain the kinetic class of the US10 gene. A mass spectrometry-based strategy was used to identify US10 in purified DEV virions and quantify its abundance.ResultsThe recombinant pET32a(+)/US10 protein was expressed as inclusion bodies, purified by gradient urea washing, and used to prepare specific antibodies. The results of qRT-PCR, western blotting, and pharmacological inhibition tests revealed that US10 is mainly transcribed in the late stage of viral replication. However, the presence of the DNA polymerase inhibitor ganciclovir and the protein synthesis inhibitor cycloheximide blocked transcription. Therefore, US10 is a γ2 (true late) gene. Indirect immunofluorescence analysis showed that US10 proteins were initially diffusely distributed throughout the cytoplasm, but with the passage of time, they gradually relocated to a perinuclear region. The US10 protein was detected in purified DEV virions by mass spectrometry, but was not detected by western blotting, indicating that DEV US10 is a minor virion protein.ConclusionsThe DEV US10 gene is a γ2 gene and the US10 protein is localized in the perinuclear region. DEV US10 is a virion component.

Preliminary study of the UL55 gene based on infectious Chinese virulent duck enteritis virus bacterial artificial chromosome clone

BackgroundLethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. However, little is known about the function of viral genes including the conserved UL55 gene among alpha herpes virus due to the obstacles in maintenance and manipulation of DEV genome in host cells.MethodsIn this paper, we constructed an infectious bacteria artificial chromosome (BAC) clone of the lethal clinical isolate duck enteritis virus Chinese virulent strain (DEV CHv) by inserting a transfer vector containing BAC mini-F sequence and selection marker EGFP into UL23 gene using homologous recombination. UL55 deletion and its revertant mutant were generated by two-step RED recombination in E. coli on basis of rescued recombinant virus. The function of UL55 gene in DEV replication and its effect on distribution of UL26.5 protein were carried out by growth characteristics and co-localization analysis.ResultsThe complete genome of DEV CHv can be stably maintained in E. coli as a BAC clone and reconstituted again in DEF cells. The generated UL55 deletion mutant based on DEV CHv-BAC-G displayed similar growth curves, plaque morphology and virus titer of its parental virus in infected Duck Embryo Fibroblast (DEF) cells. Immunofluorescence assay indicated that the loss of UL55 gene do not affect the distribution of UL26.5 protein in intracellular. These data also suggest infectious BAC clone of DEV CHv will facilitate the gene function studies of DEV genome.ConclusionsWe have successfully developed an infectious BAC clone of lethal clinical isolate DEV CHv for the first time. The generated UL55 gene mutant based on that demonstrated this platform would be a very useful tool for functional study of DEV genes. We found the least known DEV UL55 is dispensable for virus replication and UL26.5 distribution, and it could be a very promise candidate locus for developing bivalent vaccine. Experiment are now in progress for testifying the possibility of UL55 gene locus as an exogenous gene insertion site for developing DEV vectored vaccine.