Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.
Animals ; Cattle ; Dogs ; Hemagglutinins, Viral ; chemistry ; metabolism ; Influenzavirus C ; physiology ; Orthomyxoviridae Infections ; metabolism ; virology ; Protein Conformation ; Protein Folding ; Protein Processing, Post-Translational ; Viral Fusion Proteins ; chemistry ; metabolism

To investigate whether influenza virus (IFZ) could up-regulate the expression of mucin 1 (MUC1) which exists in epithelial cells of upper respiratory track to restrict the inflammation, as respiratory syncytial virus (RSV) does. Quantitative RT-PCR and Western Blot were performed to detect the expression level of MUC1 induced by two single-strand RNA viruses in A549 cell lines. HEp-2 and MDCK cells were used respectively to culture RSV and IFZ. At 24h post A549 cells infection with the same titer of RSV or IFZ, the total RNA was harvest, qRT-PCR was then performed to observe the expression level of MUC1 mRNA. Meanwhile, at 24 h and 48 h post A549 cells infection with the same titer of RSV or IFZ, the total protein and supernatant were collected respectively after cell lysis, Western Blot was then used to detect the expression level of MUC1. Results showed that RSV could up-regulate the expression of MUC1 in airway epithelial cells with a significant dose-effect correlation, whereas IFZ could not. This study firstly investigated the differences of the regulation on the expression of MUC1 induced by two single-strand RNA viruses, and demonstrated initially that the mechanism of IFZ self-limiting differed from RSV, which attributed to up-regulation of the expression level of MUC1.
Animals ; Cell Line ; Dogs ; Epithelial Cells ; metabolism ; virology ; Gene Expression Regulation ; Humans ; Influenza, Human ; genetics ; metabolism ; virology ; Madin Darby Canine Kidney Cells ; Mucin-1 ; genetics ; metabolism ; Orthomyxoviridae ; genetics ; physiology ; Respiratory Syncytial Virus Infections ; genetics ; metabolism ; virology ; Respiratory Syncytial Viruses ; genetics ; physiology ; Up-Regulation

OBJECTIVETo explore the potential effects of berberine on influenza virus infection both in vitro and in vivo.

METHODSIn vitro anti-influenza virus assays were performed by cytopathogenic effect and neuraminidase assays in Madin Darby canine kidney cells. In vivo anti-influenza virus assays were performed on the viral pneumonia model of mice. The numbers of mice that died within day 2 to day 14 postinfection were recorded to calculate the mortality. On days 2, 4, and 6, the viral titers in the lungs were determined by hemagglutination assay; hematoxylin/eosin staining was used to assess the pathogenic changes of lung tissues; the concentrations of tumor necrosis factor-alpha (TNF-α) and monocyte specific chemoattractant molecule (MCP-1) were measured by radio immunoassay or enzyme-linked immunosorbent assay; the concentrations of nitric oxide (NO) and inducible nitric oxide synthetase (iNOS) were detected by colorimetric method; reverse transcription polymerase chain reaction was used to detect the mRNA level of TNF-α and MCP-1.

RESULTSBerberine showed inhibitory effects on cytopathogenic effects and neuraminidase activity of virus, with the therapeutic index 9.69. In vivo, berberine decreased mice mortality from 90% to 55%, reduced virus titers in the lungs on day 2 postinfection (P<0.05). The lung histology scores were 1.50 ± 0.67, 4.50 ± 1.00, and 5.50 ± 1.00 in the berberine group on days 2, 4, and 6, respectively, which were significantly reduced compared to 2.17 ± 0.22, 6.83 ± 0.44, and 8.50 ± 0.33 in the infected group (P<0.05). The productions of NO and iNOS were repressed by berberine compared with those in the infected group (P<0.01). The transcription and expression of TNF-α were inhibited by berberine on day 4 (P<0.01) and day 6 (P<0.05), and those of MCP-1 were inhibited on day 6 (P<0.01) compared with the infected group.

CONCLUSIONSBerberine exhibited antiviral effects on the influenza virus both in vitro and in vivo. The possible therapeutic mechanism of berberine on influenza-induced viral pneumonia might be inhibiting the virus infection, as well as improving the pathogenic changes by repressing inflammatory substances release.

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; Berberine ; pharmacology ; therapeutic use ; Cell Line ; Chemokine CCL2 ; genetics ; metabolism ; Dogs ; Female ; Lung ; drug effects ; enzymology ; pathology ; virology ; Male ; Mice ; Neuraminidase ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; Orthomyxoviridae ; drug effects ; enzymology ; Orthomyxoviridae Infections ; complications ; drug therapy ; pathology ; virology ; Pneumonia ; complications ; drug therapy ; pathology ; virology ; Reactive Oxygen Species ; metabolism ; Survival Analysis ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Reports of influenza A virus infections in dogs has received considerable attention from veterinarians, virologists, and epidemiologists. Interaction between influenza viral hemagglutinin and cell oligosaccharides containing sialic acid residues results in infection. Sialic acids have an alpha-2,3-linkage to the penultimate galactose in the avian influenza virus receptor and an alpha-2,6-linkage in the human receptor. To date, there are no detailed data on the tissue distribution or histological features of either type of sialic acid-linked influenza virus receptors in beagle dogs, which are common laboratory animals and pets. We conducted the current study to visualize the in situ tissue distribution of both sialic acid-linked influenza virus receptors in various organs of beagle dogs using Maackia amurensis lectin II and Sambucus nigra agglutinin. Both alpha-2,3- and alpha-2,6-sialic acid-linked receptors were detected in the endothelial cells of the respiratory tract and other organs. Endothelial cells of most gastrointestinal organs were negative for alpha-2,3-sialic acid-linked receptors in the dogs. Our results suggested that these canine organs may be affected by influenza virus infection. The findings from our study will also help evaluate the occurrence and development of influenza virus infections in dogs.
Animals ; Dog Diseases/metabolism ; Dogs/metabolism/*virology ; Female ; Influenza A Virus, H5N1 Subtype/*metabolism ; Maackia/chemistry ; Male ; N-Acetylneuraminic Acid/metabolism ; Organ Specificity ; Orthomyxoviridae Infections/metabolism/transmission/veterinary ; Plant Lectins/metabolism ; Receptors, Cell Surface/analysis/chemistry/metabolism ; Receptors, Virus/analysis/chemistry/*metabolism ; Sambucus nigra/chemistry

Canine adenovirus type 2 (CAV-2) has been proposed as a vector for recombinant vaccine. Alternatively, it may be an attractive tool for gene transfer due to lack of pre-existing immunity in humans. In this study, a transfer vector based on CAV-2, in which the 1381bp fragment of the E3 region was deleted, and a linker containing the Not I, Cla I, Fse I restriction enzyme sites were cloned into the deleted region. The recombinant CAV-2 genome was released from the plasmids enzyme digestion and transfected into MDCK cells by lipofectamine to obtain the recombinant virus. No significant difference in morphology, hemagglutination and replication between the recombinant and the wide type CAV-2 was found. These results indicated that this recombinant virus CAV-2-deltaE3 (NF) may be an efficient vector for gene transfer and the capacity of the vector for inserted foreign gene was up to 3.3kb.
Adenoviruses, Canine ; genetics ; ultrastructure ; Animals ; Binding Sites ; genetics ; Cell Line ; Cloning, Molecular ; DNA Restriction Enzymes ; metabolism ; DNA, Viral ; chemistry ; genetics ; Dogs ; virology ; Genetic Vectors ; genetics ; Humans ; Lipids ; chemistry ; Microscopy, Electron ; Transfection ; methods ; Virus Replication ; genetics

The purpose of this study was to investigate the genetic features of canine coronavirus (CCV) strains detected in Korea. M gene sequences obtained for isolates from 22 dogs with enteritis over a 5-year period were evaluated. Sequence comparison revealed that the 22 Korean CCV strains had an 87.2 to 100% nucleotide homology. Comparing to the typical reference CCV strains (type II), the nucleotide sequence of Korean strains had homology ranged from 86.3% to 98.3% (89.1% to 99.2% for the amino acid sequence) and 87.7% to 97.8% (92.4% to 100% for the amino acid sequence) when compared to FCoV-like CCV strains (type I). Three amino acid variations in the M gene were characteristic for the Korean CCV strains. Phylogenetic analysis demonstrated that the 22 Korean CCV strains belonged to four typical CCV clusters (i.e., a unique Korean CCV cluster, a type II and transmissible gastroenteritis virus cluster, an intermediate cluster between type I and II, and a type I cluster). This study was the first to identify genetic differences of the M gene from Korean CCV strains and provided a platform for molecular identification of different Korean CCV strains.
Amino Acid Sequence ; Animals ; Coronavirus Infections/epidemiology/*veterinary/virology ; Coronavirus, Canine/*isolation & purification ; Dog Diseases/*epidemiology/virology ; Dogs ; Female ; Male ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/veterinary ; Republic of Korea/epidemiology ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Viral Matrix Proteins/*genetics/metabolism

Radix Isatidis (Banlangen in Chinese) is a traditional Chinese medicinal (TCM) herb, and is frequently used for treating influenza. However, the current quality control method for Radix Isatidis should be developed since it has little correlation to the pharmacodynamic action. In this paper, the in vitro inhibitory action of Radix Isatidis on neuraminidase (NA) was investigated by fluorometric assay with 4-methylumbelliferyl-D-N-acetylneuraminate (FL-MU-NANA) method. Based on the method, the experimental condition was optimized and a bioassay statistic method was established according to the reaction type and the regularity of "parallel lines of qualitative effect". Then the bioassay method of Radix Isatidis was established. This study indicated that Radix Isatidis had obvious in vitro inhibitory activity on NA with IC50 = (0.90 +/- 0.20) mg x mL(-1) (herb). The correlation between logarithmic dose and reaction rate showed an "S" shape--is quite similar to Tamiflu's reaction curve, which hinted that Radix Isatidis had the same inhibitory function on NA as Tamiflu. The established bioassay method of "parallel lines of qualitative effect" had a good reproducibility (RSD = 5.78%). The results of potency determination of Radix Isatidis were reliable (reliability test: deviation from straight line P > 0.05, deviation from parallel line P > 0.05) and well regular. As a conclusion, this bioassay method is suitable to control and evaluate the quality of Radix Isatidis.
Animals ; Antiviral Agents ; isolation & purification ; pharmacology ; Biological Assay ; Cell Line ; Dogs ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Influenza A virus ; enzymology ; Inhibitory Concentration 50 ; Isatis ; chemistry ; Kidney ; cytology ; enzymology ; virology ; Neuraminidase ; metabolism ; Oseltamivir ; pharmacology ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Quality Control ; Reproducibility of Results ; Structure-Activity Relationship