Complete Genome Sequence And Viral Characteristics Analysis Of The Hantaan Strains Isolated From Shaanxi Province

Hantaviruses (HVs) belong to the genus Bunyaviridae. They are segmented, single-stranded, negative sense RNA viruses, and like other members of the bunyavirus family, HVs are enveloped viruses with a genome that consists of three RNA segments designated L (large), M (medium), and S (small) respectively. The S segment encodes the nucleocapsid (N) protein, the M segment encodes glycoproteins of Gn and Gc, and the L segment RNA encodes RNA dependent RNA polymerase. Two diseases caused by hantaviruses, one is hemorrhagic fever with renal syndrome (HFRS) which cause severe kidney injury, and the other is hantavirus pulmonary syndrome (HPS) with symptom of non-cardiac pulmonary edema. Hantavirus infection reported annually in the world, and China is one of the most seriously epidemic countries and all reported cases are hemorrhagic fever with renal syndrome in China. From now on, there are more than 30 serotypes of hantaviruses, and Hantaan virus (Hantaan virus, HTNV) and Seoul virus (Seoul virus, SEOV) are the two main pathogens which cause HFRS. Shaanxi Province is very serious mixed viral infection area in China, and research of serological study on host animal and infection population showed the dominated prevalent strain is HTNV and Shaanxi Province has the maximum number of HFRS cases. The peak incidence appears in the winter and spring of each year, which the spring incidence from March to May is generally the onset of a small peak, while the incidence of large peak is generally concentrated in the winter, beginning from about November to the end of January the following year. Therefore, according to these data of epidemic, we chose the winter of 2014 to collect blood specimen of HFRS patients in Xi’an district.Currently, it is difficult to isolate hantavirus, most strains are isolated from animals and rare strains from human blood. In order to make more successful to isolate hantavirus, we try to isolate virus from peripheral blood mononuclear cell (PBMC), and analyze genomic features of the isolated strains, which will provide the basis of relevance of host-derived sources and HFRS patient strains for further study.The study began from November 2014 to January 2015 in Eighth Hospital in Xi’an, Shaanxi Province, we collected 38 HFRS patients’blood samples of acute phase and 29 convalescent sera. After isolation of PBMC from blood specimen and separation of sera, we inoculate the PBMC, sera and the whole blood onto the Vero-E6 cells, and viruses were detected from cultural supernatant by Real-time PCR, double-antibody sandwich ELISA and indirect immuno-fluorescent assay (IFA) after three generations of cell culture. Results showed three viral strains were successfully isolated and be conformed as Hantaan virus by Real-time PCR, two of which from PBMC and one from sera. Then sequence of three isolated strains was completed after viral RNA extraction, cDNA synthesis and segment amplification.The three new isolated strains were named as SX26, SX27 and SX30, whole genomes of the viruses was sequenced, which showed segments of S, M and L contain 1700bp, 3580bp and 6500bp respectively. After sequence alignment with the representative sequences of each viral type downloaded from GenBank and construction of phylogenetic tree with other hantaviruses, all of the nucleotide and amino acid sequences of newly isolated viruses showed high homologue with each other and with the sequence of strains XAAa10091712 isolated in Xi’an in the year of 2010. In this study, we isolated three Hantaviruses from HFRS patients’blood samples, however, we failed to isolate the SX26 and SX27 strains from peripheral blood or plasma which are from the same patients. Therefore, due to the difficulties of virus isolation and some uncontrollable factors in the process of virus isolating and culturing, we couldn’t confirm that isolate HV from PBMC samples is easier than from peripheral blood or plasma samples, but we have verified that isolate HV from PBMC samples can be seen as an effective method.To understand the serological characteristics of the new isolates, the micro neutralization test was used to do next analysis. The 50 of HFRS patients’ convalescence serum samples and the same amount of vaccine immune serum samples were neutralized by four HV strains. The result showed that all of the convalescence serum samples were identified as HTNV type, and neutralizing antibody titers of convalescence serum samples that stimulated by SX26 were higher than by Z10 strain, so the results suggested that the antigenicity of hantavirus in Xi’an area has changed in the past years. While the neutralizing antibody titers of vaccine immune serum samples that stimulated by SX26 and 84FLi strains were lower than by Z10 strain, we consider that the above results suggest that the effect of Hantavirus vaccines immunization may be reduced in this years in Xi’an area, but it still need further study.In conclusion, three strains of Hantaan viruses were isolated successfully in this study, two of them were isolated from PBMC samples, and the other one was isolated from plasma sample, and the whole genome sequences have been gained. This results may provide a scientific basis for the further study of hantaviruses epidemic strains’ genetic traits in Xi’an area. Besides, the results of serological characteristics research of HV isolates and some other representative strains laid a foundation to further evaluate the efficacy of the vaccine immunization.