No abstract available.
Coronavirus Infections/*diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/genetics/isolation & purification ; Nasopharynx/virology ; RNA, Viral/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

Avian leukosis virus subgroup J (ALV-J) is an avian retrovirus that can induce myelocytomas. A high-frequency mutation in gene envelope endows ALV-J with the potential for cross-species transmission. We wished to ascertain if the ALV-J can spread across species under selection pressure in susceptible and resistant hosts. First, we inoculated (in turn) two susceptible host birds (specific pathogen-free (SPF) chickens and turkeys). Then, we inoculated three resistant hosts (pheasants, quails and ducks) to detect the viral shedding, pathologic changes, and genetic evolution of different isolates. We found that pheasants and quails were infected under the selective pressure that accumulates stepwise in different hosts, and that ducks were not infected. Infection rates for SPF chickens and turkeys were 100% (16/16), whereas those for pheasants and quails were 37.5% (6/16) and 11.1% (3/27). Infected hosts showed immune tolerance, and inflammation and tissue damage could be seen in the liver, spleen, kidneys and cardiovascular system. Non-synonymous mutation and synonymous ratio (NS/S) analyses revealed the NS/S in hypervariable region (hr) 2 of pheasants and quails was 2.5. That finding suggested that mutation of isolates in pheasants and quails was induced by selective pressure from the resistant host, and that the hr2 region is a critical domain in cross-species transmission of ALV-J. Sequencing showed that ALV-J isolates from turkeys, pheasants and quails had moved away from the original virus, and were closer to the ALV-J prototype strain HPRS-103. However, the HPRS-103 strain cannot infect pheasants and quails, so further studies are needed.
Amino Acid Sequence ; Animals ; Avian Leukosis ; transmission ; virology ; Avian Leukosis Virus ; classification ; genetics ; physiology ; Chickens ; Ducks ; virology ; Galliformes ; virology ; Host Specificity ; Molecular Sequence Data ; Poultry Diseases ; transmission ; virology ; Quail ; virology ; Sequence Alignment ; Turkeys ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

BACKGROUND: During the 2015 outbreak of Middle East Respiratory Syndrome coronavirus (MERS-CoV), six different commercial MERS-CoV RNA detection kits based on real-time reverse-transcription polymerase chain reaction (rRT-PCR) were available in Korea. We performed analytical and clinical validations of these kits. METHODS: PowerChek (Kogene Biotech, Korea), DiaPlexQ (SolGent, Korea), Anyplex (Seegene, Korea), AccuPower (Bioneer, Korea), LightMix (Roche Molecular Diagnostics, Switzerland), and UltraFast kits (Nanobiosys, Korea) were evaluated. Limits of detection (LOD) with 95% probability values were estimated by testing 16 replicates of upstream of the envelope gene (upE) and open reading frame 1a (ORF1a) RNA transcripts. Specificity was estimated by using 28 nasopharyngeal swabs that were positive for other respiratory viruses. Clinical sensitivity was evaluated by using 18 lower respiratory specimens. The sensitivity test panel and the high inhibition panel were composed of nine specimens each, including eight and six specimens that were positive for MERS-CoV, respectively. RESULTS: The LODs for upE ranged from 21.88 to 263.03 copies/reaction, and those for ORF1a ranged from 6.92 to 128.82 copies/reaction. No cross-reactivity with other respiratory viruses was found. All six kits correctly identified 8 of 8 (100%) positive clinical specimens. Based on results from the high inhibition panel, PowerChek and AccuPower were the least sensitive to the presence of PCR inhibition. CONCLUSIONS: The overall sensitivity and specificity of all six assay systems were sufficient for diagnosing MERS-CoV infection. However, the analytical sensitivity and detection ability in specimens with PCR inhibition could be improved with the use of appropriate internal controls.
Coronavirus Infections/diagnosis/virology ; Humans ; Middle East Respiratory Syndrome Coronavirus/*genetics/isolation & purification ; Nasopharynx/virology ; Open Reading Frames/genetics ; RNA, Viral/*analysis/metabolism ; Reagent Kits, Diagnostic ; *Real-Time Polymerase Chain Reaction ; Viral Envelope Proteins/genetics

Hepatitis B virus (HBV) is the prototype of hepatotropic DNA viruses (hepadnaviruses) infecting a wide range of human and non-human hosts. Previous studies with duck hepatitis B virus (DHBV) identified duck carboxypeptidase D (dCPD) as a host specific binding partner for full-length large envelope protein, and p120 as a binding partner for several truncated versions of the large envelope protein. p120 is the P protein of duck glycine decarboxylase (dGLDC) with restricted expression in DHBV infectible tissues. Several lines of evidence suggest the importance of dCPD, and especially p120, in productive DHBV infection, although neither dCPD nor p120 cDNA could confer susceptibility to DHBV infection in any cell line. Recently, sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a binding partner for the N-terminus of HBV large envelope protein. Importantly, knock down and reconstitution experiments unequivocally demonstrated that NTCP is both necessary and sufficient for in vitro infection by HBV and hepatitis delta virus (HDV), an RNA virus using HBV envelope proteins for its transmission. What remains unclear is whether NTCP is the major HBV receptor in vivo. The fact that some HBV patients are homozygous with an NTCP mutation known to abolish its receptor function suggests the existence of NTCP-independent pathways of HBV entry. Also, NTCP very likely mediates just one step of the HBV entry process, with additional co-factors for productive HBV infection still to be discovered. NTCP offers a novel therapeutic target for the control of chronic HBV infection.
Animals ; Carboxypeptidases/genetics/*metabolism ; Gene Products, pol/genetics/metabolism ; Heparan Sulfate Proteoglycans/metabolism ; Hepatitis B virus/*physiology ; Hepatocytes/metabolism/virology ; Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors/genetics/metabolism ; RNA Interference ; Symporters/antagonists & inhibitors/genetics/metabolism ; Viral Envelope Proteins/metabolism ; Virus Internalization

To character a novel chimera(1b/2a) hepatitis C virus cell culture (HCVcc) system carrying envelope E1E2 coding gene from Hebei strain of China, chimera HCVcc (cHCVcc) was developed from Huh7.5-CD81 cells after transfection with in vitro transcribed full-length 1b/2a chimera RNA, which carrying envelope E1E2 coding gene from Hebei strain of China. Then the replication, expression and infectious titer of serial passage HCVcc were assessed by Real Time RT-PCR, indirect immunofluorescence assay (IFA) and Western blotting (WB). In addition, chimeric envelope gene from HCVcc was sequenced after serial passage. We found that the number of HCV positive focus increased gradually in cell post-transfection with chimera HCVcc (1b/2a) RNA and reach a peak platform (80% to 90%) at 41 days post-transfection; the expression of HCV protein was also confirmed by WAB during serial passage. At meantime, HCV RNA copy number in the supernatant peaked at 10(4)-10(7) copies/mL and the highest infectious titer of this 1b/2a cHCVcc reinfection were tested as 10(4) ffu/mL. Sequence analysis indicated 6 of adaptive amino acid substitutes occur among chimeric envelope E1E2 during serial passages. We con:luded that a novel 1b/2a chimera HCVcc carrying envelope E1E2 coding gene from Hebei strain of China was developed and its infectious titer increased after serial passage of HCVcc. This novel cHCVcc will be an effective tool for further evaluation of anti-virus drugs and immune effects against the major genotype from Chinese.
Cell Line ; China ; Hepacivirus ; genetics ; growth & development ; metabolism ; Hepatitis C ; virology ; Humans ; Serial Passage ; Viral Envelope Proteins ; genetics ; metabolism

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

Hepatitis c virus (HCV) infection has become one of the global public health problem,while there is no vaccine to prevent HCV infection, the so-called "cocktail" therapy that use a combination of drugs targeting multiple steps in the HCV infection cycle could achieve better curative effect. the process of HCV entering into host cell is the important step of drug intervention, in which HCV envelope protein El and E2, Host cell factors including Heparan sulfate(HS), CD81, scavenger receptor class B type I (SR-BI), Occludin (OCLD), Claudin (CLDN), low densitity lipoprotein receptor (LDLR), dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN), Liver/lymph node specific ICAM-3-grabbing integrin(L-SIGN), trans- ferrin receptor 1 (TfR1) and so on play a important role. The virus and the host factors can be used as targets of hcv entry inhibitors many studies have shown that as novel and promising compounds, HCV entry inhibitors combinating with other drugs can be more effective in the treatment of HCV, this paper have re- viewed targets and inhibitors of HCV enterring into host cell since 1990s.
Animals ; Antiviral Agents ; pharmacology ; Hepacivirus ; drug effects ; physiology ; Hepatitis C ; genetics ; metabolism ; virology ; Humans ; Receptors, Virus ; genetics ; metabolism ; Viral Envelope Proteins ; genetics ; metabolism ; Virus Internalization ; drug effects

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.
Ebolavirus ; metabolism ; Flow Cytometry ; Glycoproteins ; metabolism ; Hepatitis A Virus Cellular Receptor 1 ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Protein Binding ; Receptors, Virus ; metabolism ; Surface Plasmon Resonance ; Viral Envelope Proteins ; metabolism ; Viral Proteins ; metabolism

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

Glycoprotein N is encoded by glycoprotein N (gN) gene of herpesviruses. The amino acid composition and expression level of this protein vary among difference species of herpesviruses. According to present studies, gN protein is expressed in cytoplasm of host cells, mainly in endoplasmic reticulum. The gN forms a complex with glycoprotein M in host cells. The complex is involved in the processes of viral replication and inter-cellular infection. Moreover, this protein plays a role in immune evasion from host immune system. The study will provide a theoretical basis for further study of herpesvirus gN gene and its encoded protein.
Animals ; Herpesviridae ; genetics ; metabolism ; Herpesviridae Infections ; virology ; Humans ; Viral Envelope Proteins ; genetics ; metabolism