ABSTRACT
In order to establish a rapid and accurate method for the detection of Ebola virus (EBOV), the primers used in SYBR Green I real-time RT-PCR were designed based on the EBOV NP gene sequences published in GenBank. The SYBR Green I real-time RT-PCR was established and optimized for the detection of EBOV. The EBOV RNA that was transcribed in vitro was used as a template. The sensitivity of this method was found to reach 1.0 x 10(2) copies/microL and the detection range was 10(2) - 10(10). No cross reaction with RNA samples from Marburg virus, Dengue virus, Xinjiang hemorrhagic fever virus, Japanese encephalitis virus, Influenza virus (H1N1 and H3N2) and Porcine reproductive and respiratory syndrome virus E genomic RNA was found. The method would be useful for the detection and monitoring of EBOV in China.
Subject(s)
Humans , DNA Primers , Chemistry , Genetics , Ebolavirus , Genetics , Hemorrhagic Fever, Ebola , Virology , Organic Chemicals , Chemistry , Reverse Transcriptase Polymerase Chain Reaction , MethodsABSTRACT
ISG15 is a 15kD ubiquitin-like protein (UBL) induced by interferon (IFN). ISG15 can be covalently attached to proteins, which is called ISGylation process. ISGylation system contains ISG15, UBE1L, UBCH8 and HERC5 proteins, which are all essential for ISGylation. ISG15 and ISGylation system have been found to have anti-viral effects. A better understanding of how ISG15 mediates the anti-viral activity will provide insights for new anti-viral drugs development and new therapeutic strategies. The mechanisms underlying the ISG15 mediated anti-viral response have been explored extensively in recent years. This minireview summarized the research advances of how ISG15 mediated the anti-viral effects against different kinds of viruses.
Subject(s)
Animals , Humans , Cytokines , Physiology , HIV Infections , Allergy and Immunology , Influenza, Human , Allergy and Immunology , Ubiquitins , Physiology , Virus Diseases , Allergy and ImmunologyABSTRACT
One highly pathogenic strain of avian influenza virus (AIV) was isolated from goose in China recently, designated as F-3. In order to study the viral entry mechanisms, the hemagglutinin (HA) gene of H5N1 subtype AIV isolate was amplified by RT-PCR, and then cloned into pGEM-T vector and sequenced. The sequencing result has logging in GenBank, the accession number was AY639405. The HA gene of F-3 had a complete open reading frame (ORE) and composed of 1707 nucleotides, coding for 568 amino acids. The deduced amino acid sequence at the cleavage site of the HA protein was RKKR GLF, matched to the characteristic of virulent avian influenza strain. The HA gene were subcloned into pcDNA3, so the plasmid pcDNA-HA can express the HA glycoprotein. Co-transfected pcDNA-HA, pHIT60 (include Murine Leukemia Virus structural genes, namely gag and pol) and pHIT111 (retroviral vector genome,containing LacZ as a reporter) into 293T cells. The retroviral supernatant were harvested 48 hours post-transfection, filtered through 0.45 micromol/L filter. The supernatant were used to analysis the characteristic of the pseudotyping virions by Western blotting and infection test. Western blotting revealed the HA glycoproteins can be expressed on the virions, indicated the glycoproteins were incorporated onto the retroviral virions. Infection test were performed on 293T, NIH3T3 and COS-7, all the three kinds of cells infected were lacZ positive, indicating viral entry, and revealed the pseudotype virions of MuLV-HA were infectious. So the pseudotype system of MuLV particles with AIV Hemagglutinin proteins were setted up and it can be used to study the entry of avian influenza virus isolated from goose in China.