Identification, pathogenicity and molecular characterization of a novel fowl adenovirus 8b strain.

Since 2012, there has been a noticeable upward trend in the global incidence of inclusion body hepatitis (IBH) cases, leading to substantial economic losses in the poultry industry. In response to this trend, the current study aimed to investigate the phylogenetic information, genetic mutations, and pathogenicity of the highly pathogenic fowl adenovirus (FAdV) strain HN1472, which was isolated from liver samples obtained from a laying flock affected by IBH. This investigation was carried out using 1-day-old specific pathogen-free (SPF) chickens. Recombination and phylogenetic analyses confirmed that HN1472 is a recombinant strain derived from FAdV-8a and FAdV-8b, and exhibited significant genetic divergence in the hexon, fiber, and ORF19 genes. Notably, the phylogenetic analysis identified recombination events in these regions. Furthermore, animal experiments revealed that HN1472 is a highly pathogenic isolate, causing 80% mortality and manifesting clinical signs of IBH in SPF chickens. Furthermore, the recombinant FAdV serotype 8b (FAdV-8b) was found to be widely distributed in various tissues, with a higher concentration in the livers and gizzard tissue at 3 d postchallenge (dpc). Collectively, these findings contribute to our current understanding of the factors influencing the pathogenicity and genetic diversity of FAdV serotype 8b (FAdV-8b) in China.

Variation in the affinity of three representative avian adenoviruses for the cellular coxsackievirus and adenovirus receptor.

According to previous studies, three representative avian adenoviral strains utilize coxsackievirus-adenovirus receptor (CAR) as a receptor and seem to exhibit diverse binding affinities and modes. Thus, further revealing the exact molecular mechanism underlying the interaction between different FAdVs and the attachment receptor CAR is necessary. In this study, we successfully solved the crystal structure of the FAdV-4 fiber1 knob at 1.6 Šresolution. The interaction between the fibre knob and different domains of CAR was verified by confocal microscopy, coimmunoprecipitation and surface plasmon resonance (SPR) analysis. The fibre knobs of the three representative fowl adenoviruses specifically recognized CAR domain 1 (D1), but the recognition of CAR domain 2 (D2) by chicken embryo lethal orphan (CELO) strains was weak. These results provide insights into the differences in adenovirus‒host cell interactions and have important implications for the exploration of viral invasion mechanisms.

Development of a unique sandwich enzyme-linked immunosorbent assay based on monoclonal antibodies for the specific detection of the egg drop syndrome virus.

RESEARCH HIGHLIGHTS: A sandwich ELISA was developed to detect EDSV using the mAbs 5G4 and HRP-6G6.The sandwich ELISA maintained high specificity and sensitivity.The sandwich ELISA had equivalent consistency with real-time PCR assay.

Generation of High Concentration Free Volatile Fatty Acids from Continuous Anaerobic Digestion of Chicken Manure by Suppressing the Decomposition of Nitrogenous Components.

Free volatile fatty acids (free VFA) play a crucial role in the inactivation of pathogens during the anaerobic digestion of animal manure. However, the decomposition of nitrogenous components can release alkaline ammonium-N, which might increase the pH and reduce the concentration of free VFA. In this study, continuous anaerobic digestion of high-solid chicken manure was conducted for 150 days. The results indicated the process stabilized at a pH of approximately 6.0, with total ammonia nitrogen (TAN) of around 7.0 g/L. The resulting concentration of free VFA was only about 3.1 g/L, which might not sufficiently effective for pathogen inactivation. On the 70th day, hydrogen chloride was added into the reactor to adjust the pH to 5.5. This led to a further decrease in pH to 4.3 and TAN to 2.3 g/L. As a result, the concentration of free VFA significantly increased, reaching up to 12.6 g/L. These findings support the potential for generating high levels of free VFA even for nitrogen-rich manure by implementing an appropriate process regulation.

Type I Collagen-Adhesive and ROS-Scavenging Nanoreactors Enhanced Retinal Ganglion Cell Survival in an Experimental Optic Nerve Crush Model.

Traumatic optic neuropathy (TON) is a severe condition characterized by retinal ganglion cell (RGC) death, often leading to irreversible vision loss, and the death of RGCs is closely associated with oxidative stress. Unfortunately, effective treatment options for TON are lacking. To address this, catalase (CAT) is encapsulated in a tannic acid (TA)/poly(ethylenimine)-crosslinked hollow nanoreactor (CAT@PTP), which exhibited enhanced anchoring in the retina due to TA-collagen adhesion. The antioxidative activity of both CAT and TA synergistically eliminated reactive oxygen species (ROS) to save RGCs in the retina, thereby treating TON. In vitro experiments demonstrated that the nanoreactors preserve the enzymatic activity of CAT and exhibit high adhesion to type I collagen. The combination of CAT and TA-based nanoreactors enhanced ROS elimination while maintaining high biocompatibility. In an optic nerve crush rat model, CAT@PTP is effectively anchored to the retina via TA-collagen adhesion after a single vitreous injection, and RGCs are significantly preserved without adverse events. CAT@PTP exhibited a protective effect on retinal function. Given the abundance of collagen that exists in ocular tissues, these findings may contribute to the further application of this multifunctional nanoreactor in ocular diseases to improve therapeutic efficacy and reduce adverse effects.

Knob domain of Fiber 2 protein provides full protection against fowl adenovirus serotype 4.

Highly pathogenic fowl adenovirus serotype 4 (FAdV-4) is an acute infectious disease with severe economic impact, causing chicken hepatitis hydropericardium syndrome (HHS) and high mortality. In the present study, we evaluated the immunogenicity of the recombinant Fiber2-knob protein (F2-Knob) as an FAdV-4 candidate subunit vaccine in 14-day-old SPF chickens. The knob domain is the functional region of the viral surface protein Fiber2. The protein was expressed in Escherichia coli and was administered a single immunization with different vaccine doses. The protective efficacy was evaluated by mortality, clinical symptoms, virus shedding and histopathological examinations after challenged with the FAdV-4. The results showed that the level of ELISA antibodies of the chickens immunized with Fiber2-knob protein was significantly higher than that of the chickens immunized with an inactivated vaccine against FAdV-4. The antibody value of the immunized Fiber2-knob protein was positively correlated with the increase in immunization dose. The challenge experiment showed that the F2-Knob protein provided full protection against virulent FAdV-4 challenge and significantly reduced viral shedding. These results suggest that F2-Knob protein could be a novel vaccine candidate provide insights to control FAdV-4.

Hepatic metabolomics combined with network pharmacology to reveal the correlation between the anti-depression effect and nourishing blood effect of Angelicae Sinensis Radix.

Angelicae Sinensis Radix (AS) is reproted to exert anti-depression effect (ADE) and nourishing blood effect (NBE) in a rat model of depression. The correlation between the two therapeutic effects and its underlying mechanisms deserves further study. The current study is designed to explore the underlying mechanisms of correlation between the ADE and NBE of AS based on hepatic metabonomics, network pharmacology and molecular docking. According to metabolomics analysis, 30 metabolites involved in 11 metabolic pathways were identified as the potential metabolites for depression. Furthermore, principal component analysis and correlation analysis showed that glutathione, sphinganine, and ornithine were related to pharmacodynamics indicators including behavioral indicators and hematological indicators, indicating that metabolic pathways such as sphingolipid metabolism were involved in the ADE and NBE of AS. Then, a target-pathway network of depression and blood deficiency syndrome was constructed by network pharmacology analysis, where a total of 107 pathways were collected. Moreover, 37 active components obtained from Ultra Performance Liquid Chromatography-Triple-Time of Flight Mass Spectrometer (UPLC-Triple-TOF/MS) in AS extract that passed the filtering criteria were used for network pharmacology, where 46 targets were associated with the ADE and NBE of AS. Pathway enrichment analysis further indicated the involvement of sphingolipid metabolism in the ADE and NBE of AS. Molecular docking analysis indciated that E-ligustilide in AS extract exhibited strong binding activity with target proteins (PIK3CA and PIK3CD) in sphingolipid metabolism. Further analysis by Western blot verified that AS regulated the expression of PIK3CA and PIK3CD on sphingolipid metabolism. Our results demonstrated that sphingolipid metabolic pathway was the core mechanism of the correlation between the ADE and NBE of AS.

Membrane proteomic analysis identifies the polarity protein PARD3 as a novel antiviral protein against PEDV infection.

Porcine epidemic diarrhea virus (PEDV) is a highly pathogenic enteric coronavirus causing lethal watery diarrhea in suckling piglets. PEDV could remodel host membrane structures for their replication, assembly and escape from host cells. However, little is known about the host membrane proteins of PEDV infection. In this study, we analyzed differentially abundant proteins (DAPs) between PEDV infection group and control group and identified the polarity protein PARD3 as one of the most significantly DAPs. PARD3 is implicated in the formation of tight junctions at epithelial cell-cell contacts. Then, we found that PEDV infection promoted the degradation of PARD3 via the ubiquitin proteasome pathway. Moreover, knockdown of PARD3 promoted the proliferation of PEDV. Further study showed that the downregulation of PARD3 altered the normal morphology of the tight junction proteins and promoted apical and basolateral virus proliferation. Tight junctions enable epithelial cells to form physical barriers, which act as an innate immune mechanism that can impede viral infection and PEDV affected the barrier functions by causing degradation of PARD3. Taken together, this work is the first time to investigate the membrane protein profile of PEDV-infected cells using quantitative proteomics and suggests that PARD3 could be a potential novel antiviral protein against PEDV infection. SIGNIFICANCE: Membrane proteins are involved in various physiological and biochemical functions critical for cellular function. It is also dynamic in nature, where many proteins are changed during in response to environmental stress. However, membrane proteins are difficult to study because of their hydrophobicity. Membrane proteomic methods using mass spectrometry analysis have been developed and applied for the characterization of the plasma membrane and subcellular organelles of various virus infected cells. Porcine epidemic diarrhea virus (PEDV) is an enteric pathogen of importance to the swine industry, causing high mortality in neonatal piglets. Because PEDV infected Vero cells can lead to significant changes in cell membrane morphology and form syncytial lesions. Here, we isolated the membrane proteins of PEDV infected and control cells and applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially abundant proteins (DAPs) in PEDV-infected Vero cells and confirmed the DAPs by performing RT-qPCR and Western blot analysis. Among these differential proteins, we focused on a down-regulated protein PARD3 which is important for cell tight junction and cell polarity. Loss of PARD3 can destroy the tight junction of cells and promote the proliferation of PEDV in the apical and basolateral sides. These findings will provide valuable information to better understand the mechanisms underlying the host defense responses to PEDV infection.

Immunity analysis against Fowl Adenovirus serotype 4 (FAdV-4) based on Fiber-2 trimer Protein with the different virulence.

Since June 2015, Fowl adenovirus outbreaks have occurred in China, causing significant economic losses to poultry industry. The FAdV-4 Fiber-2 proteins could induce effective protection, but the precise mechanism of immune protection remains unknown. Here, we have compared the biological characteristics of Fiber-2 protein of the very virulent WZ strain of FAdV-4 (vvFAdV-4) with that of non-virulent ON1 strain. The sequence analysis revealed natural deletions and sequence differences between the classical non-pathogenic strain ON1 and the vvFAdV-4 isolate. These two Fiber-2 proteins successfully expressed in E. coli resemble in structure and function to the native-like trimeric protein. The trimeric structure and bioreactivity of the recombinant Fiber-2 proteins to FAdV-4 specific antibodies were characterized. The immune protection induced by Fiber-2 proteins of FAdV-4 WZ and ON1 strains were compared in SPF chickens. All birds in the WZ-Fiber-2 immunized group generated systemic specific antibodies compared with both ON1-Fiber-2 protein and PBS immunized groups. According to the results of attack mortalities, viral shedding and tissue gross lesion, the WZ Fiber-2 protein induced complete protection at a dose of 2 µg per chicken, whereas the ON1-Fiber-2 protein induced 0 protection at 3 dpc. In view of the characteristics of Fiber-2 proteins of different strains, this study can help us to further understand the mechanism of protective immunity and provide a basis for the prevention and control of FAdV-4 in chickens.

Generation and immunogenicity analysis of recombinant classical swine fever virus glycoprotein E2 and Erns expressed in baculovirus expression system.

Classical swine fever (CSF) caused by the classical swine fever virus (CSFV) is a highly contagious swine disease resulting in large economical losses worldwide. The viral envelope glycoprotein E2 and Erns are major targets for eliciting antibodies against CSFV in infected animals. In this report, the glycoprotein E2 and Erns were expressed using the baculovirus system and their protective immunity in rabbits were tested. Twenty CSFV seronegative rabbits were randomly divided into five groups. Each rabbit was intramuscularly immunized with CSFV-E2, CSFV-Erns, or their combination (CSFV-E2 + Erns). Besides, a commercial CSFV vaccine (C-strain) and PBS were used as positive or negative controls, respectively. Four weeks after the second immunization, all the rabbits were challenged with 100 RID50 of CSFV C-strain. High levels of CSFV E2-specific antibody, neutralizing antibody and cellular immune responses to CSFV were elicited in the rabbits inoculated with C-strain, CSFV-E2, and CSFV-E2 + Erns. And the rabbits inoculated with the three vaccines received complete protection against CSFV C-strain. However, no neutralizing antibody was detected in the Erns vaccinated rabbits and the rabbits exhibited fever typical of CSFV, suggesting the Erns alone is not able to induce a protective immune response. Taken together, while the Erns could not confer protection against CSFV, E2 and E2 + Erns could not only elicit humoral and cell-mediated immune responses but also confer complete protection against CSFV C-strain in rabbits.

Evaluation of chemistry and key reactor parameters for industrial water treatment applications of the UV/O3 process.

In this study, the main influence factors of combined UV/O3 process in practical industrial application were explored through laboratory trials and industrial pilot tests. Dimethyl phthalate (DMP) was analyzed as the research subject through different experiments in laboratory. The degradation effect of organic compounds by O3 and UV/O3 processes in different air distribution methods was compared independently, and the mechanism of free radical generation by the two processes was analyzed. This study found that the combined UV/O3 process for organic matter mineralization is clearly better than that of independent effect of O3 process as mixed gas-liquid distribution method was superior to the bubble aeration method. The experimental conditions included inlet O3 concentration between 70 and 75 mg/L, reactor internal relative pressure at 0.3 MPa, contact reaction time of 12 min, DMP mineralization efficiency reaching 63.07%. The calculated dosing ratio of O3 in the dynamic experiment was around 0.74 mg CODCr/mg O3. The results showed that the best effect in wastewater treatment was achieved when the conditions of ultraviolet lamp irradiation intensity and the O3 dosage reached 822.88 W/m2 15 mg/L and utilized in conjunction with biochemical reactions. The resulting CODCr concentration of effluent reached 39.8 mg/L. Finally, it is determined that the main influence factors affecting the economically efficient operation of UV/O3 process were the efficient O3 distribution mode, control of the relative pressure within the reactor, proportion of ozone addition and light source configuration.

Development of novel subunit vaccine based on truncated fiber protein of egg drop syndrome virus and its immunogenicity in chickens.

Egg-drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production and in the quality of the eggs when it infects chickens. In this report, we engineered truncated fiber protein containing the entire knob domain and part of the shaft region as a vaccine candidate. The protein was obtained in the soluble fraction in Escherichia coli (E. coli), and expression level after nickel-affinity purification was 126 mg/L. By means of multiple characterization methods, it is demonstrated that the recombinant protein retains the native trimeric structure. A single inoculation with the structure-stabilized recombinant protein, even at the lowest dose of 2 µg, stimulated hemagglutination inhibition (HI) antibody responses in chickens, for at least 16 weeks. Neutralizing titers in sera from the protein immunized groups was similar to that of inactivated vaccine immunized group. The lymphocyte proliferation response and cytokine secretion were also induced in immunized SPF chickens. In addition, immunization with the fiber protein also significantly reduced the viral load in the liver. Taken together, these results suggest the truncated fiber protein as an effective single dose, long lasting and rapidly effective vaccine to protect against EDSV.

Unravelling the receptor binding property of egg drop syndrome virus (EDSV) from the crystal structure of EDSV fiber head.

Egg drop syndrome virus (EDSV) is an avian adenovirus that causes markedly decrease in egg production, and in the quality of the eggs when it infects chickens. Until now, EDSV virus-cell interactions are poorly understood, and the cellular receptor is still unknown. In the present study, we determined the atomic structure of the fiber head of EDSV (residues 377-644) at 2.74 Šresolution. Structure comparison with the (chick embryo lethal orphan) CELO long fiber head and human adenovirus fiber heads reveals that the avian adenovirus may interact with the same attachment factor in a unique fashion. Based on the previous studies of CELO virus, we assumed that the chicken coxsackievirus and adenovirus receptor (CAR) may be the attachment factor. We then demonstrate that the chicken CAR serves as a cellular attachment factor for EDSV based on three lines of evidences. Taken together, the results presented here are helpful for further exploring the pathogenesis related to the interaction between EDSV and host cells, and may be used for vaccine development and intervention strategies against EDSV infection.

Application of antimicrobial drugs in perioperative surgical incision.

Infection in surgical incision often results in poor wound healing, and one of the main factors for wound infection is the use of antimicrobial agents. Rational use of antibiotics is one of the key factors to prevent incision infection in general surgery. The number of current clinical studies on antibiotic use before and during surgery is greater than that of systematic studies on antibiotic use after surgery. For the rational use of antibiotics and improvement of wound healing rate, researchers around the world have gradually focused on the use of antibiotics after surgery. Despite the familiarity on the concept of "rational use of antibiotics", few clear and systematic studies were conducted to elucidate the effect of different antibiotics on wound healing. Therefore, this review focuses on the use of different types of antimicrobial agents in surgical wounds.

Detection and phylogenetic analysis of porcine epidemic diarrhea virus in central China based on the ORF3 gene and the S1 gene.

BACKGROUND: Porcine epidemic diarrhea (PED) has increased in severity in China since 2010. To investigate further the infectivity, genetic diversity and molecular epidemiology of its causative agent, the porcine epidemic diarrhea virus (PEDV), we assessed 129 clinical samples, which were the intestinal tissue of piglets with severe diarrhea, from 17 cities in central China. Both the spike (S) glycoprotein (S1, 1-789 amino acids (aa)) and the full-length ORF3 gene of 21 representative field strains from 21 farms in 11 cities were sequenced and analysed. METHODS: PEDV was detected by reverse transcription-polymerase chain reaction (RT-PCR), and S1 and ORF3 sequences were processed by the Clustal W method via DNAMAN 8 software, and phylogenetic trees were constructed by the neighbor-joining method using MEGA 6 software. RESULTS: The prevalence of PEDV was 92.25% and was detected in 119 of 129 samples, with 94.03% (63 of 67) of pig farms harbouring the disease. According to the phylogenetic analysis of the S1 genes, our isolates all fell into group G2 (variants) and showed a close relationship to isolates from Chinese (HN1303, CH/ZMDZY/11 and AJ1102), Korean (AD01), American (MN, IA1, IA2 and 13-019349) sources, and these isolates differed genetically from other Chinese (LZC, CH/HNZZ/2011 and SD-M) and Korean (SM98) strains as well Japanese (83-P5 and MK) strains. In addition, our isolates differed from attenuated vaccine strains, CV777 (used in China) and DR13 (used in Korea). According to our derived amino acid sequence analysis, we detected one novel variant PEDV, viz: CH/HNLY, with 4-aa insertion/deletion (RSSS/T) at position 375 and 1-aa (D) deletion at position 430 compared to the CV777 attenuated strain. These mutations were located on the receptor binding domain. Our ORF3 gene analyses showed that the prevalent PEDV isolates were variants, and the isolated strains differed genetically from the vaccine strains. CONCLUSIONS: These findings illustrated the existence of genetic diversity among geographically distinct PEDV strains, and our study has provided an impetus to conduct further research on the PEDV receptor binding protein and on the new and efficacious vaccines design.

Cloning and Characterization of the IgA Fc Receptor from Swine.

The myeloid-specific IgA Fc receptor (FcαR) is a cell surface molecule on immunocytes that provides a fundamental connection between humoral and cellular immunity. In this study, the full-length cDNA sequence of swine FcαRI (swFcαRI) was isolated and characterized and found to contain a 792-base-pair open reading frame, encoding a 264-amino-acid transmembrane glycoprotein with a predicted molecular mass of 29.4 kDa. The swFcαRI shares high amino acid sequence homology (>50%) with its counterparts from cattle, seal, and horse. Rosetting analysis confirmed that COS-7 cells transfected with an swFcαRI expression plasmid was able to combine with chicken erythrocytes sensitized with porcine IgA, but not IgG.

High level soluble expression and one-step purification of IBDV VP2 protein in Escherichia coli.

OBJECTIVES: To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli. RESULTS: Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni-NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV. CONCLUSION: All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.