Differences in the pathogenicity and molecular characteristics of fowl adenovirus serotype 4 epidemic strains in Guangxi Province, southern China.

Starting in 2015, the widespread prevalence of hydropericardium-hepatitis syndrome (HHS) has led to considerable financial losses within China's poultry farming industry. In this study, pathogenicity assessments, whole-genome sequencing, and analyses were conducted on 10 new isolates of the novel genotype FAdV-4 during a HHS outbreak in Guangxi Province, China, from 2019 to 2020. The results indicated that strains GX2019-010 to GX2019-013 and GX2019-015 to GX2019-018 were highly virulent, while strain GX2020-019 exhibited moderate virulence. Strain GX2019-014 was characterized as a wild-type strain with low virulence, displaying no pathogenic effects when 0.5 mL containing 106 TCID50 virus was inoculated into the muscle of specific pathogen-free (SPF) chickens at 4 weeks of age, while 107 TCID50 and 108 TCID50 resulted in mortality rates of 80 and 100%, respectively. The whole genomes of strains GX2019-010 to GX2019-013, GX2019-015 to GX2019-018, and GX2020-019 showed high homology with other Chinese newly emerging highly pathogenic FAdV-4 strains, whereas GX2019-014 was closer to nonmutant strains and shared the same residues with known nonpathogenic strains (B1-7, KR5, and ON1) at positions 219AA and 380AA of the Fiber-2 protein. Our work enriches the research on prevalent strains of FAdV-4 in China, expands the knowledge on the virulence diversity of the novel genotype FAdV-4, and provides valuable reference material for further investigations into the key virulence-associated genetic loci of FAdV-4.

Biological features of fowl adenovirus serotype-4.

Fowl adenovirus serotype 4 (FAdV-4) is highly pathogenic to broilers aged 3 to 5 weeks and has caused considerable economic loss in the poultry industry worldwide. FAdV-4 is the causative agent of hydropericardium-hepatitis syndrome (HHS) or hydropericardium syndrome (HPS). The virus targets mainly the liver, and HPS symptoms are observed in infected chickens. This disease was first reported in Pakistan but has now spread worldwide, and over time, various deletions in the FAdV genome and mutations in its major structural proteins have been detected. This review provides detailed information about FAdV-4 genome organization, physiological features, epidemiology, coinfection with other viruses, and host immune suppression. Moreover, we investigated the role and functions of important structural proteins in FAdV-4 pathogenesis. Finally, the potential regulatory effects of FAdV-4 infection on ncRNAs are also discussed.

A sandwich amperometric immunosensor for the detection of fowl adenovirus group I based on bimetallic Pt/Ag nanoparticle-functionalized multiwalled carbon nanotubes.

An enzyme-free sandwich amperometric immunosensor based on bimetallic Pt/Ag nanoparticle (Pt/AgNPs)-functionalized chitosan (Chi)-modified multiwalled carbon nanotubes (MWCNTs) as dual signal amplifiers and Chi-modified MWCNTs (MWCNTs-Chi) as substrate materials was developed for ultrasensitive detection of fowl adenovirus group I (FAdV-I). MWCNTs have a large specific surface area, and many accessible active sites were formed after modification with Chi. Hence, MWCNTs-Chi, as a substrate material for modifying glassy carbon electrodes (GCEs), could immobilize more antibodies (fowl adenovirus group I-monoclonal antibody, FAdV-I/MAb). Multiple Pt/AgNPs were attached to the surface of MWCNTs-Chi to generate MWCNTs-Chi-Pt/AgNPs with high catalytic ability for the reaction of H2O2 and modified active sites for fowl adenovirus group I-polyclonal antibody (FAdV-I/PAb) binding. Amperometric i-t measurements were employed to characterize the recognizability of FAdV-I. Under optimal conditions, and the developed immunosensor exhibited a wide linear range (100.93 EID50 mL-1 to 103.43 EID50 mL-1), a low detection limit (100.67 EID50 mL-1) and good selectivity, reproducibility and stability. This immunosensor can be used in clinical sample detection.

Chicken IFI6 inhibits avian reovirus replication and affects related innate immune signaling pathways.

Interferon-alpha inducible protein 6 (IFI6) is an important interferon-stimulated gene. To date, research on IFI6 has mainly focused on human malignant tumors, virus-related diseases and autoimmune diseases. Previous studies have shown that IFI6 plays an important role in antiviral, antiapoptotic and tumor-promoting cellular functions, but few studies have focused on the structure or function of avian IFI6. Avian reovirus (ARV) is an important virus that can exert immunosuppressive effects on poultry. Preliminary studies have shown that IFI6 expression is upregulated in various tissues and organs of specific-pathogen-free chickens infected with ARV, suggesting that IFI6 plays an important role in ARV infection. To analyze the function of avian IFI6, particularly in ARV infection, the chicken IFI6 gene was cloned, a bioinformatics analysis was conducted, and the roles of IFI6 in ARV replication and the innate immune response were investigated after the overexpression or knockdown of IFI6 in vitro. The results indicated that the molecular weight of the chicken IFI6 protein was approximately 11 kDa and that its structure was similar to that of the human IFI27L1 protein. A phylogenetic tree analysis of the IFI6 amino acid sequence revealed that the evolution of mammals and birds was clearly divided into two branches. The evolutionary history and homology of chickens are similar to those of other birds. Avian IFI6 localized to the cytoplasm and was abundantly expressed in the chicken lung, intestine, pancreas, liver, spleen, glandular stomach, thymus, bursa of Fabricius and trachea. Further studies demonstrated that IFI6 overexpression in DF-1 cells inhibited ARV replication and that the inhibition of IFI6 expression promoted ARV replication. After ARV infection, IFI6 modulated the expression of various innate immunity-related factors. Notably, the expression patterns of MAVS and IFI6 were similar, and the expression patterns of IRF1 and IFN-ß were opposite to those of IFI6. The results of this study further advance the research on avian IFI6 and provide a theoretical basis for further research on the role of IFI6 in avian virus infection and innate immunity.

Follicle-stimulating hormone orchestrates glucose-stimulated insulin secretion of pancreatic islets.

Follicle-stimulating hormone (FSH) is involved in mammalian reproduction via binding to FSH receptor (FSHR). However, several studies have found that FSH and FSHR play important roles in extragonadal tissue. Here, we identified the expression of FSHR in human and mouse pancreatic islet ß-cells. Blocking FSH signaling by Fshr knock-out led to impaired glucose tolerance owing to decreased insulin secretion, while high FSH levels caused insufficient insulin secretion as well. In vitro, we found that FSH orchestrated glucose-stimulated insulin secretion (GSIS) in a bell curve manner. Mechanistically, FSH primarily activates Gαs via FSHR, promoting the cAMP/protein kinase A (PKA) and calcium pathways to stimulate GSIS, whereas high FSH levels could activate Gαi to inhibit the cAMP/PKA pathway and the amplified effect on GSIS. Our results reveal the role of FSH in regulating pancreatic islet insulin secretion and provide avenues for future clinical investigation and therapeutic strategies for postmenopausal diabetes.

A 20-Year Experience with Tissue Expansion for Large Cervical Fascial Defects: An Algorithm Based on Different Clinical Flap Designs.

Over the past 20 years, we have designed various types of expanded cervical flaps for large facial defects and achieved excellent tissue matching. This study was performed to propose a treatment strategy for flap selection for the reconstruction of different facial units. The authors retrospectively reviewed the application of cervical expanded flaps for facial rehabilitation in our department between January 2003 and January 2023. The study included 122 patients with unilateral (62.3%) and bilateral (37.7%) facial deformities ranging from the zygomatic arch to the chin. The median area of the tissue defect was 15.2 × 8.5 cm2 (ranging from 6 × 4 cm2 to 27 × 12 cm2). The expansion period ranged from 61 to 175 days (mean: 86.5 days). Maximum and minimum sizes of pre-expanded cervical flaps were 30 × 13 cm2 to 7 × 5 cm2. All the flaps could be summarized into type 1, an advanced expanded cervical flap; type 2, a wing-shaped expanded cervical flap with overlapping tissue expansion; and type 3, an expanded single-lobed transposition flap rotated based on the anterior neck. Cervical flaps reliably meet the reconstructive requirements for different facial units, especially for large cutaneous defects in the clinic. The selection of these flaps can be planned preoperatively according to the location and size of the defect or lesion.

Deep inferior epigastric vessels for free scapular flap phalloplasty.

We aimed to present our 20-year experience of using the deep inferior epigastric vessels as recipient vessels for free scapular flaps phalloplasty and evaluate the outcomes. Penile reconstruction was performed using a free scapular flap between 2000 and 2020 by the same surgical team. Deep inferior epigastric vessels were used in all the cases. The surgical techniques and outcomes were described. Overall, 73 patients used the deep inferior epigastric artery (DIEA) as the recipient artery. Regarding the recipient veins, 2 veins were anastomosed in 72 (98.6%) patients, 1 deep inferior epigastric vein (DIEV) was used in 1 patient, 2 DIEV in 14, 1 DIEV + superficial inferior epigastric vein (SIEV) in 13, 1 DIEV + superficial circumflex iliac vein (SCIV) in 38, great saphenous vein (GSV) + SCIV in 4, and GSV + SIEV in 3. The mean age and body mass index of the study cohort was 28 years and 24.3 kg/m2, respectively. The shortest follow-up time was 7 months. Eleven patients had flap-related complications. Three patients were readmitted to the operating room within 24 hours, and 2 of them underwent salvage procedures with venous revision. Two patients lost the entire flap. One patient with 3-cm distal portion necrosis required surgical intervention. Three patients experienced urethral necrosis. DIEA is a suitable receptor artery for inflow. The DIEV, SIEV, and SCIV are available options for venous drainage according to the patient anatomical characteristics. The GSV can be an excellent backup for outflow and salvage procedures.

Pathogenicity and molecular characteristics of fowl adenovirus serotype 4 with moderate virulence in Guangxi Province, China.

The GX2020-019 strain of fowl adenovirus serotype 4 (FAdV-4) was isolated from the liver of chickens with hydropericardium hepatitis syndrome in Guangxi Province, China, and was purified by plaque assay three times. Pathogenicity studies showed that GX2020-019 can cause typical FAdV-4 pathology, such as hydropericardium syndrome and liver yellowing and swelling. Four-week-old specific pathogen-free (SPF) chickens inoculated with the virus at doses of 103 median tissue culture infectious dose (TCID50), 104 TCID50, 105 TCID50, 106 TCID50, and 107 TCID50 had mortality rates of 0, 20, 60, 100, and 100%, respectively, which were lower than those of chickens inoculated with other highly pathogenic Chinese isolates, indicating that GX2020-019 is a moderately virulent strain. Persistent shedding occurred through the oral and cloacal routes for up to 35 days postinfection. The viral infection caused severe pathological damage to the liver, kidney, lung, bursa of Fabricius, thymus, and spleen. The damage to the liver and immune organs could not be fully restored 21 days after infection, which continued to affect the immune function of chickens. Whole genome analysis indicated that the strain belonged to the FAdV-C group, serotype 4, and had 99.7-100% homology with recent FAdV-4 strains isolated from China. However, the amino acid sequences encoded by ORF30 and ORF49 are identical to the sequences found in nonpathogenic strains, and none of the 32 amino acid mutation sites that appeared in other Chinese isolates were found. Our research expands understanding of the pathogenicity of FAdV-4 and provides a reference for further studies.

Blood Supply of the Temporal Flap Pedicled With Orbicularis Oculi Muscle: Anatomy and Its Clinical Implications.

BACKGROUND: Traumatic injury or tumor resection can lead to eyelid defects, nasal defects, and cheek defects. The temporal flap pedicled with orbicularis oculi muscle (OOM) can be used to repair these defects. This cadaver-based anatomic study aimed to evaluate the blood supply of this flap and investigate its clinical implications. METHODS: Twenty hemifaces from 10 cadavers were used in this study. The number of arteries supplying OOM of the flap, the diameter of the artery entering OOM, and the maximum width of OOM were recorded. All data were presented as mean±SD values and analyzed using Student t -test. A P value<0.05 was considered statistically significant. RESULTS: Of these 10 specimens, 7 were males and 3 were females. The average age was 67.7 years (range, 53-78 y). The number of arteries supplying OOM was 8.5±1.4 in the male and 7.8±1.2 in the female. The diameter of the zygomatico-orbital artery was detected as 0.53±0.06 mm in the male and 0.40±0.11 mm in the female. The maximum width of OOM was detected as 2.5±0.1 cm in the male and 2.2±0.1 cm in the female. Males had significantly larger average values than females in the diameter of zygomatico-orbital artery and maximum width of OOM ( P =0.012, P <0.001, respectively). However, the number of arteries supplying OOM did not differ significantly between sex ( P =0.322). CONCLUSIONS: We conclude that the blood supply of the temporal flap pedicled with OOM is abundant and reliable. The findings provide surgeons with valuable anatomic knowledge for repairing facial defects with this flap.

Using an Expanded Cervical Flap With an Overlapping Tissue Expansion Technique to Resurface Middle and Lower Facial Defects.

BACKGROUND: Extensive facial burn scars are a tragedy for patients and often pose a great challenge to surgeons because of the high esthetic and functional demands. For patients with healthy skin in the neck region, a cervical flap is highly recommended for facial resurfacing; however, the skin on the midline of the neck often needs more expansion than that on either side, especially for the treatment of large facial defects. The sufficient longitudinal soft tissue in the anterior neck ensures a normal neck shape as well as a normal range of cervical extension, rotation, and lateral flexion. To overcome this, we developed an expanded cervical flap with an overlapping tissue expansion technique to gain more length centrally. METHODS: First, 2 tissue expanders were embedded in the anterior neck region overlapping each other at the midline of the neck. After adequate inflation of the expander, the expanded flap was dissected and rotated to repair defects in the middle and lower face. The anchor position of the flap was placed on the horizontal line of the thyroid cartilage to restore the cervicomental angle. RESULTS: Sixteen patients were treated with this method in this single-center study. All defects affected the middle and lower face, with an area ranging from 135 to 185 cm 2 , and were caused by a massive facial burn. Among them, 12 patients suffered ectropion of the lower lip, 3 suffered limited mouth opening due to scar contraction, and one patient had a cervicomental adhesion. The area of the expanded flap was approximately 163 to 266 cm 2 . The average period of expansion was 89.5 days. Patients were followed up after the operation, with the follow-up period ranging from 6 to 12 months. In all cases, good defect coverage was achieved, with primary closure of the donor sites and a good postoperative cervical configuration. CONCLUSION: We conclude that the expanded cervical flap with the overlapping tissue expansion technique proved to be a reliable method for facial skin reconstruction with functional and aesthetic improvement.

A multiplex fluorescence-based loop-mediated isothermal amplification assay for identifying chicken parvovirus, chicken infectious anaemia virus, and fowl aviadenovirus serotype 4.

Chicken parvovirus (ChPV), chicken infectious anaemia virus (CIAV) and fowl adenovirus serotype 4 (FAdV-4) are avian viruses that have emerged in recent years and have endangered the global poultry industry, causing great economic loss. In this study, a multiplex fluorescence-based loop-mediated isothermal amplification (mLAMP) assay for detecting ChPV, CIAV and FAdV-4 was developed to simultaneously diagnose single and mixed infections in chickens. Three primer sets and composite probes were designed according to the conserved regions of the NS gene of ChPV, VP1 gene of CIAV and hexon gene of FAdV-4. Each composite probe was labelled with a different fluorophore, which was detached to release the fluorescence signal after amplification. The target viruses were distinguished based on the colour of the mLAMP products. The mLAMP assay was shown to be sensitive, with detection limits of 307 copies of recombinant plasmids containing the ChPV target genes, 749 copies of CIAV and 648 copies of FAdV-4. The assay exhibited good specificity and no cross-reactivity with other symptomatically related avian viruses. When used on field materials, the results of the mLAMP assay were in 100% agreement with those of the previously published PCR assay. The mLAMP assay is rapid, economical, sensitive and specific, and the results of amplification are directly observable by eye. Therefore, the mLAMP assay is a useful tool for the clinical detection of ChPV, CIAV and FAdV-4 and can be applied in rural areas.

Explore how immobilization strategies affected immunosensor performance by comparing four methods for antibody immobilization on electrode surfaces.

Among the common methods used for antibody immobilization on electrode surfaces, which is the best available option for immunosensor fabrication? To answer this question, we first used graphene-chitosan-Au/Pt nanoparticle (G-Chi-Au/PtNP) nanocomposites to modify a gold electrode (GE). Second, avian reovirus monoclonal antibody (ARV/MAb) was immobilized on the GE surface by using four common methods, which included glutaraldehyde (Glu), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS), direct incubation or cysteamine hydrochloride (CH). Third, the electrodes were incubated with bovine serum albumin, four different avian reovirus (ARV) immunosensors were obtained. Last, the four ARV immunosensors were used to detect ARV. The results showed that the ARV immunosensors immobilized via Glu, EDC/NHS, direct incubation or CH showed detection limits of 100.63 EID50 mL-1, 100.48 EID50 mL-1, 100.37 EID50 mL-1 and 100.46 EID50 mL-1 ARV (S/N = 3) and quantification limits of 101.15 EID50 mL-1, and 101.00 EID50 mL-1, 100.89 EID50 mL-1 and 100.98 EID50 mL-1 ARV (S/N = 10), respectively, while the linear range of the immunosensor immobilized via CH (0-105.82 EID50 mL-1 ARV) was 10 times broader than that of the immunosensor immobilized via direct incubation (0-104.82 EID50 mL-1 ARV) and 100 times broader than those of the immunosensors immobilized via Glu (0-103.82 EID50 mL-1 ARV) or EDC/NHS (0-103.82 EID50 mL-1 ARV). And the four immunosensors showed excellent selectivity, reproducibility and stability.

An Enzyme-Free Sandwich Amperometry-Type Immunosensor Based on Au/Pt Nanoparticle-Functionalized Graphene for the Rapid Detection of Avian Influenza Virus H9 Subtype.

Avian influenza virus H9 subtype (AIV H9) has contributed to enormous economic losses. Effective diagnosis is key to controlling the spread of AIV H9. In this study, a nonenzymatic highly electrocatalytic material was prepared using chitosan (Chi)-modified graphene sheet (GS)-functionalized Au/Pt nanoparticles (GS-Chi-Au/Pt), followed by the construction of a novel enzyme-free sandwich electrochemical immunosensor for the detection of AIV H9 using GS-Chi-Au/Pt and graphene-chitosan (GS-Chi) nanocomposites as a nonenzymatic highly electrocatalytic material and a substrate material to immobilize capture antibodies (avian influenza virus H9-monoclonal antibody, AIV H9/MAb), respectively. GS, which has a large specific surface area and many accessible active sites, permitted multiple Au/Pt nanoparticles to be attached to its surface, resulting in substantially improved conductivity and catalytic ability. Au/Pt nanoparticles can provide modified active sites for avian influenza virus H9-polyclonal antibody (AIV H9/PAb) immobilization as signal labels. Upon establishing the electrocatalytic activity of Au/Pt nanoparticles on graphene towards hydrogen peroxide (H2O2) reduction for signal amplification and optimizing the experimental parameters, we developed an AIV H9 electrochemical immunosensor, which showed a wide linear range from 101.37 EID50 mL-1 to 106.37 EID50 mL-1 and a detection limit of 100.82 EID50 mL-1. This sandwich electrochemical immunosensor also exhibited high selectivity, reproducibility and stability.

Facial Defects With Infraorbital and Zygomatic Area Reconstruction Using an Expanded Flap Based on the Orbicularis Oculi Muscle: A Long-Term Follow-up.

OBJECTIVE: Reconstruction of facial soft-tissue defects may pose a dilemma for plastic surgeons, as the flaps must be reliable to obtain a natural appearance while minimizing donor site morbidities. This clinical study describes a reconstructive method for infraorbital and zygomatic defects using a pre-expanded rotation flap based on the orbicularis oculi muscle (OOM). METHODS: The surgeries were subdivided into 2 stages. In the first stage of the operation, a 100 to 200 mL expander was placed underneath the temporal area through a hairline incision. In the second stage, after adequate inflation of the expander, the pre-expanded rotation flap based on the OOM of the lower eyelid was raised from lateral to medial to cover the facial defects. RESULTS: In this single-center study from February 2010 to February 2017, 16 patients underwent facial defect reconstruction using the pre-expanded flap based on the OOM. All of the defects were located at the infraorbital and zygomatic regions, and their sizes ranged from 3.0 4.0 to 7.0 14.0 cm. The causes of these defects included postburn scars (37.5%), melanocytic nevus (50%), and hemangiomas (12.5%). In all cases, good coverage was provided for the defects that were in the medial cheek or lower eyelids. There were no flap losses of any kind. There were no major complications, and all minor incidences were treated by minimal procedures. The patients were followed up after surgery, with the follow up ranging from 6 months to 108 months. The follow-up data included postoperative consultations, the defect size, the need for further procedures and the degree of satisfaction. CONCLUSION: The pre-expanded rotation flaps in the lateral facial area based on the OOM can ideally and safely be applied for facial defect reconstruction owing to their reliable blood supply and excellent texture match.

Facial and Neck Reconstruction With Pre-expanded Medial Upper Arm Flap: An Alternative method and 20-Year Experience.

PURPOSE: To present our experience with pre-expanded medial upper arm flap in facial and neck reconstruction. PATIENTS AND METHODS: This was a retrospective study operated between January 1st, 2001 and January 1st, 2021, at the Plastic Surgery Hospital, Chinese Academy of Medical Sciences, and Peking Union Medical College. Staged face and/or neck reconstruction was performed. RESULTS: Forty-one patients were treated in our institution and thirty-eight patients (forty-three flaps) were included in this cohort as. They ranged from 6 to 44 years old. There was no total flap loss in the cohort. Partial flap necrosis was observed in the earlier patients (4 cases). CONCLUSION: Pre-expanded medial upper arm flap is well matched to the facial and neck skin in color, texture, and thickness. Considering the excellent aesthetic outcomes, this flap is a good alternative for selected patients with soft tissue defects of the head and neck.

Integrated Multi-Omics Analysis Reveals the Effect of Maternal Gestational Diabetes on Fetal Mouse Hippocampi.

Growing evidence suggests that adverse intrauterine environments could affect the long-term health of offspring. Recent evidence indicates that gestational diabetes mellitus (GDM) is associated with neurocognitive changes in offspring. However, the mechanism remains unclear. Using a GDM mouse model, we collected hippocampi, the structure critical to cognitive processes, for electron microscopy, methylome and transcriptome analyses. Reduced representation bisulfite sequencing (RRBS) and RNA-seq in the GDM fetal hippocampi showed altered methylated modification and differentially expressed genes enriched in common pathways involved in neural synapse organization and signal transmission. We further collected fetal mice brains for metabolome analysis and found that in GDM fetal brains, the metabolites displayed significant changes, in addition to directly inducing cognitive dysfunction, some of which are important to methylation status such as betaine, fumaric acid, L-methionine, succinic acid, 5-methyltetrahydrofolic acid, and S-adenosylmethionine (SAM). These results suggest that GDM affects metabolites in fetal mice brains and further affects hippocampal DNA methylation and gene regulation involved in cognition, which is a potential mechanism for the adverse neurocognitive effects of GDM in offspring.

Intrauterine Hyperglycemia Alters the Metabolomic Profile in Fetal Mouse Pancreas in a Gender-Specific Manner.

Mounting evidence has shown that intrauterine hyperglycemia exposure during critical stages of development may be contributing to the increasing prevalence of diabetes. However, little is known about the mechanisms responsible for offspring metabolic disorder. In this present study, we explored intrauterine hyperglycemia exposure on fetal pancreatic metabolome, and its potential link to impaired glucose tolerance in adult offspring. Here, using a GDM mouse model, we found the metabolome profiling of pancreas from male and female fetus showing altered metabolites in several important pathways, including 5-methylcytosine, α-KG, branched-chain amino acids, and cystine, which are associated with epigenetic modification, insulin secretion, and intracellular redox status, respectively. This finding suggests that intrauterine exposure to hyperglycemia could cause altered metabolome in pancreas, which might be a metabolism-mediated mechanism for GDM-induced intergenerational diabetes predisposition.

Gallus NME/NM23 nucleoside diphosphate kinase 2 interacts with viral σA and affects the replication of avian reovirus.

Our present study aimed to identify host cell proteins that may interact with avian reovirus (ARV) σA protein and their potential effect on ARV replication. The ARV structural protein σA has been demonstrated to suppress interferon production and confirmed to activate the PI3K/Akt pathway. However, host cell factors interacting with σA to affect ARV replication remain unknown. In current study, a cDNA library of chicken embryo fibroblasts (CEFs) was constructed, and host cell proteins interacting with σA were screened by a yeast two-hybrid system. We identified four candidate cellular proteins that interact with ARV σA protein. Among them, Gallus NME/NM23 nucleoside diphosphate kinase 2 (NME2) was further validated as a σA-binding protein through co-immunoprecipitation. The key interaction domain was identified at amino acids (aa) 121-416 in NME2 and at aa 71-139 in σA, respectively. We demonstrated that overexpression of NME2 substantially inhibited ARV replication. In addition silencing NME2 by small interfering RNAs (siRNAs) resulted in marked enhancement of ARV replication. Our work has demonstrated that NME2 is a σA-binding protein that may affect ARV replication in CEF cells.

Genetic characterization of fowl aviadenovirus 4 isolates from Guangxi, China, during 2017-2019.

Hepatitis-hydropericardium syndrome (HHS) is a severe disease that causes 20 to 80% mortality in chickens aged 3 to 6 wk. Fowl aviadenovirus serotype 4 (FAdV-4) plays an important role in the etiology of HHS. Since 2015, outbreaks of HHS have been reported in several provinces of China; however, details regarding the FAdV-4 genome properties are lacking. In the present study, the complete genomes of 9 isolates responsible for these outbreaks in Guangxi Province, China, were sequenced. To investigate the molecular characteristics of these FAdV-4 isolates, we compared their genomes with those of other reported pathogenic and nonpathogenic FAdV-4 isolates. A variable number of GA repeats were observed in the isolates of this study. Each of the isolates GX2017-01, GX2017-02, GX2018-08, and GX2019-09 had 11 GA repeats; GX2017-03, GX2017-04, and GX2017-05 each had 10 GA repeats, while GX2017-06 and GX2018-07 each had 8 GA repeats. We observed several deletions and distinct amino acid mutations in the major structural genes of these isolates when compared with non-Chinese isolates. We found 2 novel putative genetic markers in the hexon protein, one present in GX2017-02, in which aspartic acid (D) was changed to tyrosine (Y), and another present in each of isolates GX2018-08 and GX2019-09, in which serine (S) was changed to arginine (R), when compared with selected Chinese and some non-Chinese isolates. Moreover, the phylogenetic analysis revealed that all the isolates of this study were clustered within FAdV-C. We found that these isolates were closely related to other recently isolated Chinese strains. The data presented in this study will not only increase the understanding of the molecular epidemiology and genetic diversity of FAdV-4 isolates in China but also has an important reference value of the major factors that determine the virulence of FAdV-4 strains.

Electrochemical immunosensor with Cu(I)/Cu(II)-chitosan-graphene nanocomposite-based signal amplification for the detection of newcastle disease virus.

An electrochemical immunoassay for the ultrasensitive detection of Newcastle disease virus (NDV) was developed using graphene and chitosan-conjugated Cu(I)/Cu(II) (Cu(I)/Cu(II)-Chi-Gra) for signal amplification. Graphene (Gra) was used for both the conjugation of an anti-Newcastle disease virus monoclonal antibody (MAb/NDV) and the immobilization of anti-Newcastle disease virus polyclonal antibodies (PAb/NDV). Cu(I)/Cu(II) was selected as an electroactive probe, immobilized on a chitosan-graphene (Chi-Gra) hybrid material, and detected by differential pulse voltammetry (DPV) after a sandwich-type immune response. Because Gra had a large surface area, many antibodies were loaded onto the electrochemical immunosensor to effectively increase the electrical signal. Additionally, the introduction of Gra significantly increased the loading amount of electroactive probes (Cu(I)/Cu(II)), and the electrical signal was further amplified. Cu(I)/Cu(II) and Cu(I)/Cu(II)-Chi-Gra were compared in detail to characterize the signal amplification ability of this platform. The results showed that this immunosensor exhibited excellent analytical performance in the detection of NDV in the concentration range of 100.13 to 105.13 EID50/0.1 mL, and it had a detection limit of 100.68 EID50/0.1 mL, which was calculated based on a signal-to-noise (S/N) ratio of 3. The resulting immunosensor also exhibited high sensitivity, good reproducibility and acceptable stability.