We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R²=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.
Animals ; Reproducibility of Results ; Influenza A Virus, H7N9 Subtype ; Influenza in Birds ; Chromatography, Gel ; Virion ; Lasers

Ebola virus (EBOV) is an enveloped negative-sense RNA virus and a member of the filovirus family. Nucleoprotein (NP) expression alone leads to the formation of inclusion bodies (IBs), which are critical for viral RNA synthesis. The matrix protein, VP40, not only plays a critical role in virus assembly/budding, but also can regulate transcription and replication of the viral genome. However, the molecular mechanism by which VP40 regulates viral RNA synthesis and virion assembly/budding is unknown. Here, we show that within IBs the N-terminus of NP recruits VP40 and is required for VLP-containing NP release. Furthermore, we find four point mutations (L692A, P697A, P698A and W699A) within the C-terminal hydrophobic core of NP result in a stronger VP40-NP interaction within IBs, sequestering VP40 within IBs, reducing VP40-VLP egress, abolishing the incorporation of NC-like structures into VP40-VLP, and inhibiting viral RNA synthesis, suggesting that the interaction of N-terminus of NP with VP40 induces a conformational change in the C-terminus of NP. Consequently, the C-terminal hydrophobic core of NP is exposed and binds VP40, thereby inhibiting RNA synthesis and initiating virion assembly/budding.
Ebolavirus/physiology* ; HEK293 Cells ; HeLa Cells ; Humans ; Nucleocapsid Proteins/metabolism* ; RNA, Viral/metabolism* ; Viral Matrix Proteins/metabolism* ; Virion/metabolism* ; Virus Assembly

virion pretreatment with trypsin or neuraminidase. Additionally, the HA assay results showed a significant positive correlation with the infectious viral titer. Our results suggest that assessing the HA activity of PDCoV may be a useful diagnostic method for investigating and surveilling PDCoV infections.]]>
Coronavirus ; Diarrhea ; Erythrocytes ; Hemagglutination ; Methods ; Neuraminidase ; Population Characteristics ; Swine ; Trypsin ; Virion

virion protection against neutralization by antibodies. In infected cattle, BoHV-4 induces an immune response characterized by low neutralizing antibody levels or an absence of such antibodies. Therefore, virus seroneutralization in vitro cannot always be easily demonstrated. The aim of this study was to evaluate the neutralizing capacity of 2 Argentine BoHV-4 strains and to associate those findings with the gene expression profiles of the major envelope glycoproteins. Expression of genes coding for the envelope glycoproteins occurred earlier in cells infected with isolate 10/154 than in cells infected with strain 07/435, demonstrating a distinct difference between the strains. Differences in serological response can be attributed to differences in the expression of antigenic proteins or to post-translational modifications that mask neutralizing epitopes. Strain 07/435 induced significantly high titers of neutralizing antibodies in several animal species in addition to bovines. The most relevant serological differences were observed in adult animals. This is the first comprehensive analysis of the expression kinetics of genes coding for BoHV-4 glycoproteins in 2 Argentine strains (genotypes 1 and 2). The results further elucidate the BoHV-4 life cycle and may also help determine the genetic variability of the strains circulating in Argentina.]]>
Adult ; Animals ; Antibodies ; Antibodies, Neutralizing ; Argentina ; Cattle ; Clinical Coding ; Epitopes ; Glycoproteins ; Glycosylation ; Humans ; In Vitro Techniques ; Kinetics ; Life Cycle Stages ; Masks ; Protein Processing, Post-Translational ; Transcriptome ; Virion

Influenza vaccine-associated anaphylaxis is a very rare allergic reaction to vaccines, but the most concerning and life-threatening adverse reaction. Although the safety of influenza vaccines has been well documented, occasional cases of anaphylaxis in vaccinated patients have been reported. In this study, we analyzed the immunoglobulin E (IgE) response to whole influenza vaccines in a pediatric case of delayed-onset anaphylaxis after influenza vaccination. The patient showed elevated specific IgE levels against whole influenza vaccines, especially with split virion from egg-based manufacturing process. Specific IgE levels to influenza vaccines showed decreased over. We evaluated a causal relationship between influenza vaccine and anaphylaxis event by enzyme-linked immunosorbent assay. Delayed-onset anaphylaxis after influenza vaccination can occur in children without predisposing allergic diseases. In addition, the results suggested that formulation and production system of influenza vaccines could affect the probability of severe allergic reaction to vaccines.
Anaphylaxis ; Child ; Drug Hypersensitivity ; Enzyme-Linked Immunosorbent Assay ; Humans ; Hypersensitivity ; Hypersensitivity, Delayed ; Immunoglobulin E ; Immunoglobulins ; Influenza Vaccines ; Influenza, Human ; Vaccination ; Vaccines ; Virion

Chronic hepatitis B (CHB) infection leads to clinically heterogeneous disease outcomes. Different viral markers are utilized to monitor treatment effects and predict risk of complications in patients with CHB. Hepatitis B core-related antigen (HBcrAg) is a novel serum composite viral protein whose performance has been proven to be superior to that of existing viral markers. It showed good correlation with intrahepatic covalently closed-circular DNA. Its profile differs drastically in patients in different disease phases, and the level declines with antiviral therapies. HBcrAg may be helpful for predicting hepatocellular carcinoma development and hepatitis B virus (HBV) reactivation in immunosuppressed patients. Another emerging measurable serum marker, HBV RNA, exists in the form of pregenomic RNA-containing virions. Its profile differs between patients in different disease phases in a similar manner to that of HBcrAg. HBV RNA is present in serum at lower levels than HBV DNA in treatment-naive patients by 1–2 logs. In contrast, its level is higher than HBV DNA in patients receiving nucleos(t)ide analogues (NAs). A significant decline in serum RNA was observed in patients receiving novel antiviral therapies, including core protein allosteric modulators and RIG-1/NOD2 agonists. Both HBcrAg and HBV RNA may be helpful for predicting off-therapy sustained virological control in patients who stop long-term NA treatment.
Biomarkers ; Carcinoma, Hepatocellular ; DNA ; Hepatitis B ; Hepatitis B virus ; Hepatitis B, Chronic ; Hepatitis, Chronic ; Humans ; RNA ; Virion

Type 2 diabetes mellitus has become one of the fastest growing public health problems worldwide. The disease is believed to involve a complex process involving genetic susceptibility and environmental factors. The human intestine harbors hundreds of trillions of bacteria, as well as bacteriophage particles, viruses, fungi, and archaea, which constitute a complex and dynamic ecosystem referred to as the gut microbiota. Increasing evidence has indicated changes in the gut microbiota composition or function in type 2 diabetic patients. An analysis of ‘dysbiosis’ enables the detection of alterations in the specific bacteria, clusters of bacteria, or bacterial functions associated with the occurrence of type 2 diabetes. These bacteria are involved predominantly in the control of inflammation and energy homeostasis. This review attempts to show that the gut microbiota are important factors for the occurrence of type 2 diabetes and are important for the treatment of gut microbiota dysbiosis through bariatric surgery, fecal microbiota transplantation, prebiotics, and probiotics.
Archaea ; Bacteria ; Bacteriophages ; Bariatric Surgery ; Diabetes Mellitus, Type 2 ; Dysbiosis ; Ecosystem ; Fecal Microbiota Transplantation ; Fungi ; Gastrointestinal Microbiome ; Genetic Predisposition to Disease ; Homeostasis ; Humans ; Inflammation ; Intestines ; Prebiotics ; Probiotics ; Public Health ; Virion

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.
Agaricales ; Chromatography ; Fruit ; Influenza A virus ; N-Acetylneuraminic Acid ; Neuraminidase ; Sialic Acids ; Silica Gel ; Virion

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.
Adenoviridae* ; Adenoviruses, Human ; Biology ; Codon, Initiator ; Genetic Therapy ; Genome ; Genome, Viral* ; Leucine Zippers ; Species Specificity ; Transfection* ; Virion*

Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.
Animals ; Baculoviridae ; Blotting, Western ; Clone Cells ; Cloning, Organism ; Enzyme-Linked Immunosorbent Assay ; Influenza, Human* ; Insects ; Mice ; Microscopy, Electron, Transmission ; Toxoplasma* ; Toxoplasmosis ; Virion