Hongyan Chen

Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein

Abstract S1D (residues 636–789) is a neutralizing epitope region on the spike protein (S) of porcine epidemic diarrhea virus (PEDV). To accurately identify epitopes on S1D, the S1-phage library containing the gene encoding the S1D region of PEDV S protein was micropanned by six specific monoclonal antibodies (McAbs) against the S1D region. These micropanned epitope regions (MER) were focused on 696–779 amino acids of the S protein. To further map epitopes of the MER, seven overlapping mini-fragments covering MER nucleotides were separately synthesized and expressed in Escherichia coli BL21 with a GST tag. These mini-GST fusion proteins were scanned by ELISA and Western blotting with the six McAbs, and the result showed that S1D5 (residues 744–759) and S1D6 (residues 756–771) are two linear epitopes of the PEDV S protein. The antisera of the epitopes S1D5 and S1D6 could react with the native S protein of PEDV. Furthermore, Pepscan of the two linear epitopes demonstrated that SS2 (748YSNIGVCK755) and SS6 (764LQDGQVKI771) are two core epitopes on S1D5 and S1D6, respectively, located on the S protein of PEDV.

Autophagy activated by duck enteritis virus infection positively affects its replication

Duck enteritis virus (DEV) is an acute, septic, sexually transmitted disease that occurs in ducks, geese and other poultry. Autophagy is an evolutionarily ancient pathway that is important in many viral infections. Despite extensive study, the interplay between DEV and autophagy of host cells is not clearly understood. In this study, we found that DEV infection triggers autophagy in duck embryo fibroblast (DEF) cells, as demonstrated by the appearance of autophagosome-like double- or single-membrane vesicles in the cytoplasm of host cells and the number of GFP-LC3 dots. In addition, increased conversion of the autophagy marker protein LC3-I and LC3-II and decreased p62/SQSTM1 indicated complete autophagy flux. Heat-inactivated DEV infection did not induce autophagy, suggesting that the trigger of autophagy in DEF cells depended on DEV replication. When autophagy was pharmacologically inhibited by LY294002 or wortmannin, DEV replication decreased. The DEV offspring yield decreased when small interference RNA was used to interfere with autophagy related to the genes Beclin-1 and ATG5. In contrast, after treating DEF cells with rapamycin, an inducer of autophagy, DEV replication increased. These results indicated that DEV infection induced autophagy in DEF cells and autophagy facilitated DEV replication.

Impaired Cellular Energy Metabolism Contributes to Duck-Enteritis-Virus-Induced Autophagy via the AMPK–TSC2–MTOR Signaling Pathway

Duck enteritis virus (DEV) is a large, complex double-stranded DNA virus that induces duck embryo fibroblast (DEF) cells autophagy, which is beneficial to its own replication, but the mechanism has not been described. In this study, we showed that impaired cell energy metabolism is involved in DEV-induced autophagy, whereby ATP synthesis is disrupted in cells after DEV infection, which causes metabolic stress and activation of autophagy. Methyl pyruvate (MP) inhibited conversion of LC3I to LC3II and accumulation of GFP-LC3, which could reverse the energy loss caused by DEV infection. Inhibition of DEV replication by MP confirmed the above view. We found that infection with DEV activated the metabolic regulator 5′ AMP-activated kinase (AMPK) and inhibited activity of mechanistic target of rapamycin (mTOR). In the cases where AMPK expression was inhibited, the LC3-I conversion to LC3-II ratio was decreased, as was GFP-LC3 point and DEV replication; in addition, inhibition of p-mTOR showed a reverse trend. We also found that tuberous sclerosis (TSC) 2 was a key mediator between AMPK and mTOR through activation by phosphorylation. siRNA targeting TSC2 was transfected to reverse the inhibition of mTOR, and down-regulate autophagy level and DEV replication, but AMPK expression was not changed, while siRNA targeting AMPK inhibited activation of TSC2. In conclusion, our findings indicate that energy metabolism in cell damage induced by DEV contributes to autophagy via the AMPK–TSC2–MTOR signaling pathway, which provides a new perspective for DEV and host interactions.

L-Glutamine Supplementation Alleviates Constipation during Late Gestation of Mini Sows by Modifying the Microbiota Composition in Feces

Constipation occurs frequently in both sows and humans, particularly, during late gestation. The microbial community of the porcine gut, the enteric microbiota, plays a critical role in functions that sustain intestinal health. Hence, microbial regulation during pregnancy may be important to prevent host constipation. The present study was conducted to determine whether L-glutamine (Gln) supplementation improved intestinal function and alleviated constipation by regulation of enteric microbiota. 16S rRNA sequences obtained from fecal samples from 9 constipated sows (3 in the constipation group and 6 in the 1.0% Gln group) were assessed from gestational day 70 to 84. Comparative analysis showed that the abundance of intestinal-friendly microbiota, that is, Bacteroidetes (P = 0.007) and Actinobacteria (P = 0.037), was comparatively increased in the 1.0% Gln group, while the abundance of pernicious bacteria, Oscillospira (P < 0.001) and Treponema (P = 0.011), was decreased. Dietary supplementation with 1.0% Gln may ameliorate constipation of sows by regulated endogenous gut microbiota.

Selection of an aptamer against Muscovy duck parvovirus for highly sensitive rapid visual detection by label-free aptasensor

Muscovy duck parvovirus (MDPV) causes high mortality and morbidity in ducks. This study investigated a novel aptamer-based, label-free aptasensor detection of MDPV. In this study, we developed an ssDNA aptamer using the filtration partition and lambda exonuclease method with an affinity-based monitor and counter-screening process. After 15 rounds of SELEX (systematic evolution of ligands by exponential enrichment), the ssDNA aptamer Apt-10, which specifically bound to MDPV with high affinity (Kd = 467nM) was successfully screened, and the aptamer was also found to be good specific to MDPV. The selected Apt-10 aptamer can be used to distinguish MDPV and goose parvovirus (GPV). Three-dimensional structural analysis of the Apt-10 aptamer indicated that it folded into a compact stem-loop motif, which was related to its high affinity. Finally, a label-free detection method based on unmodified gold nanoparticles and Apt-10 aptamer was developed for MDPV determination. The concentration of Apt-10 aptamer at 5μM was optimal for MDPV determination in the label-free aptasensor. Excellent linearity was acquired and the lowest detection limit was 1.5 or 3 EID50 (50% egg infection dose) of MDPV, respectively, depending upon spectrophotometry or the naked eye were used. These results show the potential of the aptamer for the rapid detection of MDPV and antiviral research.

Rapid and sensitive detection of mink circovirus by recombinase polymerase amplification

To date, the pathogenic role of mink circovirus (MiCV) remains unclear, and its prevalence and economic importance are unknown. Therefore, a rapid and sensitive molecular diagnosis is necessary for disease management and epidemiological surveillance. However, only PCR methods can identify MiCV infection at present. In this study, we developed a nested PCR and established a novel recombinase polymerase amplification (RPA) assay for MiCV detection. Sensitivity analysis showed that the detection limit of nested PCR and RPA assay was 101 copies/reaction, and these methods were more sensitive than conventional PCR, which has a detection limit of 105 copies/reaction. The RPA assay had no cross-reactivity with other related viral pathogens, and amplification was completed in less than 20 min with a simple device. Further assessment of clinical samples showed that the two assays were accurate in identifying positive and negative conventional PCR samples. The detection rate of MiCV by the RPA assay in clinical samples was 38.09%, which was 97% consistent with that by the nested PCR. The developed nested PCR is a highly sensitive tool for practical use, and the RPA assay is a simple, sensitive, and potential alternative method for rapid and accurate MiCV diagnosis.

DEV induce autophagy via the endoplasmic reticulum stress related unfolded protein response

Duck enteritis virus (DEV) can infect ducks, geese, and many other poultry species and leads to acute, septic and highly fatal infectious disease. Autophagy is an evolutionarily ancient pathway that plays an important role in many viral infections. We previously reported that DEV infection induces autophagy for its own benefit, but how this occurs remains unclear. In this study, endoplasmic reticulum (ER) stress was triggered by DEV infection, as demonstrated by the increased expression of the ER stress marker glucose-regulated protein 78 (GRP78) and the dilated morphology of the ER. Pathways associated with the unfolded protein response (UPR), including the PKR-like ER protein kinase (PERK) and inositol-requiring enzyme 1 (IRE1) pathways, but not the activating transcription factor 6 (ATF6) pathway, were activated in DEV-infected duck embryo fibroblast (DEF) cells. In addition, the knockdown of both PERK and IRE1 by small interfering RNAs (siRNAs) reduced the level of LC3-II and viral yields, which suggested that the PERK-eukaryotic initiation factor 2α (eIF2α) and IRE1-x-box protein1 (XBP1) pathways may contribute to DEV-induced autophagy. Collectively, these data offer new insight into the mechanisms of DEV -induced autophagy through activation of the ER stress-related UPR pathway.

Selection of a DNA aptamer against zearalenone and docking analysis for highly sensitive rapid visual detection with a label-free aptasensor

Contamination of feed with zearalenone (ZEN) presents a significant risk to animal health. Here, a visible, rapid, and cost-effective aptamer-based method is described for the detection of ZEN. After 8 rounds of SELEX (systematic evolution of ligands by exponential enrichment) with an affinity-based monitor and counter-screening process, the ssDNA aptamer Z100 was obtained, which had high affinity (dissociation constant = 15.2 ± 3.4 nM) and good specificity. Docking analysis of Z100 indicated that noncovalent bonds (π-π interactions, hydrogen bonds, and hydrophobic interactions) helped ZEN to anchor in the binding sites. Finally, a label-free detection method based on gold nanoparticles and Z100 at 0.25 μM was developed for ZEN determination. Excellent linearity was achieved, and the lowest detection limit was 12.5 nM. This rapid and simple method for ZEN analysis has high sensitivity and can be applied for on-site detection of ZEN in animal feeds.

Application of Real-Time Quantitative PCR to Detect Mink Circovirus in Naturally and Experimentally Infected Minks

The mink circovirus (MiCV), a newly discovered pathogen, is associated with diarrhea in farmed minks. The prevalence and economic importance of this virus remain poorly understood, and a quantitative method for diagnosis of MiCV infection has not been established. This research aims to develop a highly specific, sensitive, and quantitative assay for MiCV. A Real-Time quantitative polymerase chain reaction (qPCR) assay was developed to detect different isolates of the MiCV in mink samples. The qPCR system is highly sensitive with a detection limit of as low as 10 viral DNA copies. The specificity of this qPCR assay was supported by the absence of cross-reaction with other pathogens. The coefficients of variation were low for both inter-assay and intra-assay variabilities. In addition, the results also expressed the distribution of MiCV in infectious mink tissues with high levels of virus in the skeletal muscle and heart. The heart occupied a higher proportion than other tissues, which can be considered the primary source of test material. This qPCR method could be a useful tool for epidemiological studies and disease management. This method for MiCV is highly specific, sensitive, repeatable, quantitative, and can rapidly determine viral load levels in different tissues samples.

Lactobacillus brevis 23017 Relieves Mercury Toxicity in the Colon by Modulation of Oxidative Stress and Inflammation Through the Interplay of MAPK and NF-κB Signaling Cascades

Aims: Lactobacillus strains have protective effects against heavy metals while relieving oxidative stress and modulating the immune response. Mechanisms that ameliorate heavy metal toxicity and the relationship between probiotics and gut barrier protection in the process of heavy metal pathogenesis was poorly understood. Methods and Results: In this study, Lactobacillus brevis 23017 (LAB, L. brevis 23017), a selected probiotics strain with strong mercury binding capacities, was applied to evaluate the efficiency against mercury toxicity in a mouse model. Histopathological results along with HE stains show that L. brevis 23017 protects the integrity of the small intestinal villus, which slows weight loss in response to Hg exposure. The qRT-PCR results demonstrate that L. brevis 23017 maintains a normal mucosal barrier via modulation of tight junction proteins. Importantly, the present study demonstrates that L. brevis 23017 effectively ameliorates injury of the small intestine by reducing intestinal inflammation and alleviating oxidative stress in animal models. Moreover, L. brevis 23017 blocks oxidative stress and inflammation through MAPK and NF-κB pathways, as shown by western blot. Conclusions: Together, these results reveal that L. brevis 23017 may have applications in the prevention and treatment of oral Hg exposure with fermented functional foods by protecting gut health in daily life.