Study On Hantavirus Gene Characteristics And Construction Of Pseudovirus In Hubei Province

Aims:This project aims to investigate the phylogenetic characterization of hantaviruses currently circulating in edemic area of Hubei Province. Furthermore, construction of hantaviruses pseudovirions based on different types of hantaviruses provide a new tool to study the functional characteristics of hantaviral glycoproteins.Methods:1. During the years of2009to2011, rodents were captured from HTC, YJW, DLM, AJB, SP and ZJW and HFRS patients’sera were collected as well. The positive samples were screened by means of IFA (indirect immunofluorescence assay), FRNT (focus reduction neutralization test) and RT-PCR.2. A pair of specific primer was designed for typing the hantavirus subtypes, and all the products of RT-PCR were cloned into the pGEM-T vectors for sequencing. Phylogenetic and homologous analysis based on the partial hantavirus S gene were determined by MEGA.3. Sucking BALB/c was applied for isolation of hantavirus by serial passages. The S, M, and L-segments of the isolates were amplified by specific primers and whole-genome sequences were analyzed by software of bioinformatics.4. A vesicular stomatitis virus (VSV) pseudotype bearing SNV, ANDV, HTNV and PHV envelope glycoproteins were produced. The morphology and titration of these pseudotypes were determined.5. The vesicular stomatitis virus (VSV) pseudotype was applied in HFRS/HPS patients’ sera and monoclonal antibody neutralization test as a comparison for live hantavirus. Meanwhile, the way that VSV pseudotype enter the cells was observed by specific receptor inhibitor.6. Rescue of recombinant VSV bearing chimeric glycoproteins. which contained glycoproteins of pathogenic and nonpathogenic hantavirus (GPC genes either using the Gn gene of SNV fused to the Gc gene of PHV (pSNVMPHV) or the Gn gene of PHV fused to the Gc gene of SNV (pPHVMSNV)). Analyzed the antigenicity of chimeric pseudovirions using patients’sera.7. The GPs of HV004strain was applied to construct the VSV pseudotype. And the reactivity of HV004VSV pseudotype and specific antibody was detected by flow cytometry. Arbidol was added before the HV004VSV pseudotype infected the monolayer Vero-E6to observe the inhibitory activities.Results:1. A total of173rodents including48Apodemus agrarius and125Rattus norvegicus were captured from six trapping sites in Jiangxia District. Hantavirus nucleotides were detected in three Apodemus agrarius and four Rattus norvegicus. The detective rate of hantavirus nucleotides in the rodent species is7/173(4.05%) in Jiangxia District. Besides, specific neutralizing antibodies of SEOV or HTNV were detected in10of11serum samples of suspected HFRS patients (hantavirus nelcetide acid were detected in two serum samples).2. Based on sequences recovered from all the positive hantaviral samples, the phylogenectic analysis showed that both the HTN and SEO hantavirus were the predominant hantavirus subtypes in Jiangxia District. SEOV strains including the JX1143, JX0937, JX1018and JX1028identified in Jiangxia fell into the cluster with Z37and ZT10, while the the partial S segment of hantavirus amplified from two sera (s200903and s201012) was found to have a high identity to the counterpart of the hantavirus strain (HV003, HV004and HV005) from A. Agrarius, which was confirmed to be HTNV subtype, falling into a new lineage.3. HV004was successfully isolated by passage in newborn mice. The complete S, M, and L-segments of HV004were determined and compared with other hantaviruses. Phylogenetic analysis showed that HV004belonged to the Hantaan virus(?)HTNV). but it did not fit into any other lineages of HTNV we have known vet. The differences between strain HV004and other HTNV (76-118,A9,CGHu2,CGRn45and Q32strains) were83%-90%at nucleotide level and96%-99%at amino acid level. Several different deduced amino acids were found in some sites of HV004compared with the other HTNV. During the process of isolation in the model of sucking BALB/c mice, only one nucleotide substitution occurred in the non-coding region in the third passage but without changes of pathogenicity.4. The titers of VSV pseudotype was ranged from the3×105IU/ml (VSV△G-ANDV-GFP) to2X10’IU/ml (VSV△G-VSV-GFP). Under the observation of electron microscopy, as expected, the particles appeared very similar to but less long than wild-type VSV, with a bullet-shaped morphology. For VSV△G-luc-HTNV, the average length of the bullet-shaped particles was163.7±10.7nm and width of71.1±6.6nm.5. Same inhibitory effects as live hantaviruses were observed when hantavirus pseudotypes reacted with monoclonal antibodies against Gc of HTNV, mAb VSV, serum of patients infected with SNV, ANDV and HTNV.6. Anti-SNV serum could react with VSV bearing chimeric envelop glycoproteins, and the neutralizing effect was between the SNV pseudotypes and PHV pseudotypes. And neutralizating reaction to PHV pseudotypes was better than the SNV pseudotypes.7. ReoPro, a kind of β3integrin inhibitor, could block the live pathogenic hantavirus entry the Vero-E6cells and the same effect was also observed with pseudovirions. Furthermore, the UV-inactivated the SNV and HTNV could inhibt the SNV and HTNV pseodotypes entry cells separately.8. Arbidol had the specialty of inhibiting the hantavirus pseudotypes entry the Vero-E6cells. HV004hantavirus pseudotypes could be appied for high throughput flow (?)ytometry that maybe utilized to rapidly screen antihantavirus drugs. Conclusion:1. A new subtype of Hantaan virus was discovered circulating in edemic area of Hubei province and it was probablely be responsible for some recent HFRS cases2. The characterization that hantavirus pseudotypes could model the live hantaviruses in aspects of entry and antigenicity makes pseudovirions can be applied to investigate interaction between hantaviral glycoproteins and cell, tropism and substitute of native hantavirus to screen drugs...