ABSTRACT
In order to detect the nucleic acid of Puumala hantavirus, RNA was extracted from lungs of bank voles captured in Northeast China, and partial S and M genome segments of Puumala virus were amplified by RT-PCR and sequenced. Phylogenetic analysis suggested that Chinese Puumala virus had diverged from the common node of PUUV, with accumulating nucleotide substitutions and formed a distinct lineage from other Puumala viruses. Newly found Puumala virus was most closely related to the Kamiiso-8Cr-95 and Tobetsu-60Cr-93 strains which came from Japan and the muju strains which came from South Korea. By analysis of S and M genome segments of Puumala virus, we deduced a new Puumala virus subtype did exist in Northeast China.
Subject(s)
Animals , Base Sequence , Chlorocebus aethiops , China , Evolution, Molecular , Genome, Viral , Orthohantavirus , Genetics , Molecular Sequence Data , Phylogeny , Puumala virus , Classification , Genetics , Reverse Transcriptase Polymerase Chain Reaction , Rodentia , Virology , Sequence Analysis, DNA , Vero CellsABSTRACT
To improve the rescue efficiency of measles virus cDNA clone, the cell line that stably expressed the T7 RNA polymerase was established. Firstly, the T7 RNA polymerase gene was amplified by PCR and then the PCR product was inserted into pcDNA3 to obtain plasmid pcDNA3-T7. Vero cell was transfected with the plasmid and G418 was added to the cell 24h later to kill the cells without the plasmid. Western blotting analysis showed that the Vero/pcDNA3-T7 cell could express T7 RNA polymerase. To analyze the gene function of T7 RNA polymerase, the pT7IP-EGFP plasmid was transfected into the Vero/pcDNA3 T7 cell and EGFP was analized by fluorescence. The result suggested that T7 RNA polymerase expressed in the Vero/pcDNA3-T7 cell could transcribe the gene under control of the T7 promoter. Moreover, the minigenome PminiEGFP inserted reversely with report gene EGFP was established. After trans fection with the plasmid and infection with measles virus, EGFP was expressed, indicating the Vero/pcDNA3-T7 cell could rescue the minigenome.
Subject(s)
Animals , Blotting, Western , Cell Line , Chlorocebus aethiops , DNA-Directed RNA Polymerases , Genetics , Metabolism , Green Fluorescent Proteins , Genetics , Metabolism , Measles virus , Genetics , Microscopy, Fluorescence , Recombinant Fusion Proteins , Genetics , Metabolism , Transfection , Vero Cells , Viral Proteins , Genetics , Metabolism , Virus ReplicationABSTRACT
<p><b>BACKGROUND</b>To study the full length L and M sequence of Hantavirus Q32 strain gene and explore its molecular characters.</p><p><b>METHODS</b>The L and M segment cDNA of Hantavirus Q32 strain was amplified by RT-PCR. The purified PCR products were sequenced directly or cloned into pGEM-T Vector and then sequenced.</p><p><b>RESULTS</b>The L genome segment of Q32 virus was found to be 6533 nucleotides in length. One large open reading frame was found located at bases 38 to 6493. This was predicted to encode an L protein 2151 amino acids in length with a molecular mass of 2.46 x 10(5). The M genome segment was 3616 nucleotides in length. One open reading frame was located at bases 41 to 3488. This was predicted to encode an M protein 1135 amino acids with a molecular mass of 1.26 x 10(5).</p><p><b>CONCLUSION</b>The nucleotides sequence of M and L segments of strain Q32 was similar to that of other Hantavirus M and L segments. Deduced amino acid sequences of glycoprotein and RNA polymerase revealed high homologue to other Hantavirus.</p>