Genetic Analysis Of Hantaviruses And Their Hosts In Hubei Province

ObjectiveHantaan virus (HTNV) and Seoul virus (SEOV) are two major zoonotic pathogens of hemorrhagic fever with renal syndrome (HFRS) in Asia. Hubei province had been one of the most severe epidemic areas of HFRS. During 2000 to 2009, the incidence and mortality of HFRS cases has declined in Hubei. To find out what cause the changing of epidemiological landscape in last decade. Systematic evolution and molecular epidemiology of HTNV, SEOV in their hosts were carried out in Hubei province. The research demonstrates the prevalence and distribution of these viruses and indicates the epidemiologic tendencies, therefore worthy for the prevention and control of HFRS.Methods1. From 2000 to 2009, rodents were trapped in four HFRS epidemic areas of Hubei province, China: southwest mountains (Dangyang), central hills (Nanzhang), Jianghan plain (Xinzhou and Jiangxia), and southeast low hills (Qichun).2. Colletion and screening of the serum samples from patients with HFRS in Hubei. Serum samples from patients with HFRS were collected during 1986-1987 and 2000-2009 at the teaching hospitals of Hubei Medical University. All the samples had been screened by clinical diagnosis and the detection of specifie IgM against hantaviruses by ELISA.3. Lung tissues were screened by (IFA) to detect hantavirus antigens. Total RNA was extracted from rodent lung tissues and the serum samples from patients by using the RNAprep Tissue Kit.4. The primers for S- and M-segments were designed according to HTNV and SEOV sequences obtained through GenBank.5. Total DNA was extracted from lung tissues using the DNAprep Tissue Kit. The primers for the region of the D-loop of mitochondrial DNA were designed according to the sequences obtained through GenBank.6. RT-PCR was performed to study the variability of partial S. M gene of hantaviruses isolated from the samples. Furthermore, PCR was performed to study variability of partial D-loop of mice which infected with Hantanvirus. The positive productions of RT-PCR, PCR were recovered, purification and sequenced by the Invitrogen Company in Shanghai.7. Multiple sequence alignment was carried out using BioEdit 7.0 and ClustalX 2.0 with default parameters. The occurrence of recombination events among hantaviruses sequences were evaluated using multiple recombination-detection methods, including RDP, GENECONV, MaxChi, Chimaero and 3seq implemented in the RDP3 Beta 4.9 program. The Akaike information criterion implemented in Modeltest 3.7 were used to estimate the most suitable model of nucleotide substitution. Phylogenetic trees were constructed using Bayesian Markov chain Monte Carlo (BMCMC) implemented in MrBayes 3.1 and Neighbor-joining analyses in PAUP* 4.0.8. For the partial S and M segment sequences of hantavirus variants, estimates of genetic divergence were obtained by pairwise analysis using MEGA 4.1. All positions containing gaps and missing data were eliminated from the dataset. Number of alleles, allele diversity, and nucleotide diversity were calculated with Arlequin 3.11 and DnaSP 5.00. The null assumptions of neutrality and constant population size were tested with Tajima's D test, Fu's Fs test and R2 test.Results1. Between 2000 and 2009, a total of 687 rodents including 148 (21.54%) A. agrarius.507 (73.80%) R. norvegicus and 32 (4.66%) R. tanezumi were captured from five trapping sites in Hubei province. Rattus norvegicus were the predominant rodent species. 2. Lung tissues were screened by (IFA) to detect hantavirus antigens, and 46 (6.70%) samples, including 11/148 (7.43%) A agrarius,35/507 (6.90%) R. norvegicus and none of R. tanezumi were identified positive. The total percentages of positive rodents were similar among all the locations, ranging from 6.30% to 7.62%. Whereas, the positive percentage rates of A. agrarius and R. norvegicus varied among different sampling sites.3. Partial S segment sequence analysis indicated that among the 46 hantavirus positive samples,15.22%(7/46) were HTNV, and 84.78%(39/46) were SEOV variants. SEOV was the predominant hantaviruses genotype among rodents. We first found that SEOV caused spillover infections to A. agrarius naturally.4. Phylogenetic analyses of partial S segment sequences of SEOV variants identified from rodents in Hubei fell into four lineages, including three previously undefined lineages(SEO-S-#7～SEO-S-#9). HTNV variants from Hubei fell into one established lineage (HTN-S-#7) and a novel lineage (HTN-S-#10) based on partial S segment sequence. And most of SEOV was similar with Z37 which isolated from Zhejiang province. HTNV obtained in the study was high homology with 76-119, Q32.5. In the phylogenetic tree based on partial M segment sequences of SEOVs, the variants identified from rodents in Hubei fell into five lineages. The clustering patterns of lineages SEO-M-#3 and#8 were similar to those of S segment sequences. However, clustering patterns of lineages of SEO-M-#7,#9 and #10 were different, suggesting inter-lineage reassortment occurred among SEOV variants. The partial M-segment sequences of these variants also clustered in the same pattern.6. The phylogenesis showed the virus-positive rodents has small genetic distance with virus-negative rodents and confirmed that 4 SEOV detected in the study were amplied from the lungs of Apodemus agrarius.7. Phylogenetic analyses of partial S segment amplified from serum samples of patients with HFRS in Hubei during 1986-1987 and 2000-2009 showed that virus strains fell into SEOV and HTNV. Most SEOVs was similar with Z37 which isolated from Zhejiang province, and HTNV obtained from patients in the study was high homology with 76-118.Conclusion1. Molecular analysis of hantavirus sequences from patients during 1986-1987 and 2000-2009 were high homology. The increased proportion of R. norvegicus in rodents and their widespread distribution led to the prevalence of SEOV in recent years, which may be an important reason for the declined incidence and mortality of HFRS in China.2. Inter-lineage reassortment and spillover infection to A. agrarius were observed for the SEOV variants due to the frequent inter- and intra- species interactions among rodents, suggested that evolution of this virus was not as steady as previously thought.3. There are at least six phylogenetic lineages of SEOVs in Hubei, including three already known lineages (#1,#3 and #4) and three novel lineages (#7-#9). It is suggested Hubei province is an important epidemic focus of SEOV with relatively high divergence of the virus.The abundance of the phylogenetic lineages of SEOV suggested that central-south China was a radiation center for SEOVs.