Establishment Of Avian Influenza Virus And Hantavirus Reverse Genetics System

Avian Influenza (AI) is a serious zoonotic disease caused by Type A Avian Influenza Virus (AIV) of the family Orthomyxoviridae. By June 19,2008, totally 385 persons were infected by this virus, and 243 of them died. This disease caused huge economic losses, and public health and social security issues. In recent years, the newly developed reverse genetics technique of AIV provides an important platform for the study on AIV. We constructed a bi-direction transcription plasmid pZL2006 by amplifying human RNA polymeraseâ… promoter sequence from Hela cells, synthesizing RNA polymeraseâ… terminator sequence, and inserting the combinations of promter and terminator sequence into eukaryotic expression plasmid pVAX1. The constructed plasmid pZL2006 contained two sets of promoter and termination sequences, namely RNA polymeraseâ…¡promoter (CMV promoter) and termination sequences (aâ…¡BGH), RNA polymeraseâ… promoter (Pâ… h) and murine polymeraseâ… terminator sequences (tâ… ). The positive cDNA encoding vRNA was inserted between polymeraseâ…¡promoter and termination sequence to achieve vRNA transcription and viral protein expression. In this study, we amplified the complete genome sequence of H5N1 strain A/Viet Nam/1194/2004(H5N1) isolated from Vietnam in 2004, and obtained eight gene fragments. The eight gene fragments were sequenced and their sequences compositions matched those in GenBank. These fragments were inserted into vector pZL2006, which was confirmed by sequencing positive clones from restriction digestion. The eight recombinant plasmids containing the complete gene fragment of AIV constructed the reverse genetics system of AIV. The eight-plasmid system was transfected into the 293 T/MDCK cells by using Liposome 2000. After 36 h of this transfection, most of the infected cells became round, died, and small pieces of cells fell off etc, while the control cell group was in normal state. The spherical particle, about 80-120 nm in diameter, having capsule surface with spike was observed in the supernatant of transfected cells with eight plamids by electron microscope, which was characterisitic morphology of influenza viron. The results of hemagglutination test (HA) showed that the titer to rescue virus was between 26 to 27, which means that the rescued virus has hemagglutinin activity and a higher titer. After 36 h of reinoculation of the rescued virus in MDCK cells, the infected cells became round, died, small pieces of cells fell off etc., which were very similar to the cytopathic characteristics of MDCK cells infected by the wild strain. After 48 h of the inoculation of the supernatant of rescued virus in 10 chicken embryo eggs, all of them died, while the control chicken embryo eggs survived. This indicated that both the recombinant virus and the wild virus have lethality to chicken embryo eggs. PCR amplification and sequencing confirmed that the rescued virus is identical to the A/Viet Nam/1194/2004(H5N1) strain in the sequence composition from the same region. Based on the experimental results mentioned above, it can be concluded that the reverse genetics operating system of AIV was successfully constructed in this study.Hantaviruses belonging to Hantavirus, Bunyaviridae, can cause human hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, and hantavirus pulmonary syndrome (HPS) in America. HFRS is the most serious in China, and more than 90 percent cases were reported in our country. Hantavirus strain H8205 was isolated in this lab from the serum of one HFRS patient in Heilongjiang province by using Vero-E6 cells. This strain can produce strong immunological responses to the serum of HFRS patients from most of China. The NP of this strain was used as antigen, and was very useful in the diagnosis of Hantavirus prevalent in China. The biological characteristics and animal pathogenic test were investigated in this study, and a series of results were obtained, but the complete genome has not been sequenced. Currently, the sequences of the genes L, M, S of Hantavirus H8205 Strain including the complete 5' and 3'ends were obtained by using RT-PCR and RACE. Compared with the published sequences of Hantavirus representative strains, H8205 has 95.5%-98.3% amino acid homology and 81.9%-88.8% nucleic acid homology in L gene,85.2%-99.7% amino acid homology and 74.5%-99.3% nucleic acid homology in M gene, and 97.2%-99.8% amino acid homology and 83.5%-99.4% nucleic acid homology in S gene. The phylogenetic tree based on the L/M/S gene sequence analysis indicated that H8205 is genetically closer to other strains belonging to the same Hantavirus type, while distantly related to DOBV, SEOV, SNV, PUUV. By reference strains, we can find similar strains from different countries or regions, which provides information for the further study on the differences among strains and their antigen variation.Hantavirus is negative-sense RNA virus. RNA structure characteristics and instability makes it very difficult to operate the genome of RNA virus at the molecular level. The successful establishment of reverse genetics operating system of segmented negative-sense RNA virus, e.g. AIV, is a new start point for the study of Influenza Virus. However, the construction of Hantavirus reverse genetics systems has not established yet, which prevented from performing the related studies of Hantavirus due to lacking an important study platform. In this study, the Hantavirus H8205 strain was chosen. The complete gene fragments of this strain were obtained by segment amplification. The gene fragments were ligated into the full-length cDNA of L/M genes by usingâ…¡s restriction endonucleases. The sequencing results showed that the obtained full-length cDNA was completely correct, without any mutations or deletions. Two recombinant plasmids, PCDNA-L/PCDNA-S, were constructed by inserting the L/S genes of H8205 into the eukaryotic expression vector pcDNA-3.1(+)/pcDNA-3.1-Hygro(+). Two trans-acting proteins L and NP of Hantavirus were expressed by transfecting eukaryotic cells with the RNA polymeraseâ…¡promoter (PCMV) and terminator sequences in the eukaryotic expression vector. The plasmid pp2006 containing human RNA polymerase I promoter and murine RNA polymeraseâ… terminator was constructed. The non-coding region of Hantavirus L gene and GFP fusion gene were inserted between RNA polymerase I promoter (Pâ… h) and the terminator (Tâ… ) to form a transcription unit. After the transfection into cells, the fusion gene containing the 5'and 3'non-coding regions of L gene of Hantavirus and GFP coding gene, was expressed by using the polymerase I in cells and the promoter Pâ… h. The trans-acting protein of Hantavirus combined the fusion gene into RNPs and the expression of GFP protein was activated. After 24 h of the transfection of Hantavirus subgenomic infectious clone (PCDNA-L/PCDNA-S/ PP2006-L-GFP) into the 293 T cells by using Liposome 2000, obvious green fluorescent protein was observed in the transfected cells, while there is no green fluorescent protein observed in the control cells. This means that the subgenomic infectious clone of Hantavirus was successfully established, which is an important part to establish the reverse genetics system of Hantavirus. The system of subgenome can be used to evaluate RNA transcription efficiency of the plasmid containing RNA polymerase I, and find the optimal transfection conditions and optimized the constructed plasmid by detecting the expression quantity of the reporter gene GFP, which will lay a solid foundation for the final establishment of Hantavirus reverse genetics system.