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Objective:To establish a double antibody sandwich ELISA for detecting the specific antigen of Seoul virus (SEOV) L99 strain and to provide a means for antigen detection in the development, production and verification of vaccine against hemorrhagic fever with renal syndrome (HFRS).Methods:Monoclonal antibodies (McAbs) aganist L99 virus were induced in mice using four hybridoma cell lines and purified by Protein-A affinity chromatography. The purity, titer and specificity of McAbs were determined by SDS-PAGE, indirect ELISA and Western blot, respectively. Four McAbs were paired with each other and the additivity indices of paired McAbs were analyzed. After labeling McAbs with horseradish peroxidase (HRP), the concentrations of the coated and labeled antibodies were optimized by orthogonal test, and then a double antibody sandwich ELISA for virus antigen detection was established. Type Ⅱ HFRS inactivated vaccine standard was used as a quantitative standard to verify the sensitivity, linearity, specificity, accuracy and precision of the developed method. The applicability of the method was verified by testing three batches of vaccine stock solutions.Results:Four McAbs were at titers of greater than 1∶10 6 and their purity was all greater than 98%. The McAbs secreted by 1D5, 3A4 and 5B7 cells could specifically recognize the nucleocapsid protein of SEOV L99. There was cross-reaction between McAb secreted by 1D5 cells and Hantaan virus PS-6. The McAbs secreted by 3A4 and 1D5 were used as coating and labeling antibodies based on the results of antibody pairs. The working concentrations of the coating antibody and the horseradish peroxidase (HRP)-labeled antibody were 20 μg/ml and 1∶4 000, respectively. The minimum detection limit of the established method for the detection of SEOV L99 antigen was 0.078 1 μg/ml, and the linear range was 0.078 1-2.500 0 μg/ml with a R2 value of more than 0.99. There was no cross reaction with other HFRS vaccine. The virus antigen recovery rate was between 95.8% and 108.7%, and the coefficients of variation of precision was less than 10%. Three batches of Type II HFRS inactivated vaccine stocks were detected by this method and the results was dose-dependent. Conclusions:This study successfully established a double antibody sandwich ELISA method for specific detection of SEOV L99 strain antigen in the production of bivalent HFRS vaccines produced from hamster kidney cells.
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Objective Genetic analysis was performed to infer the relationship between hantaviruses carried by Rattus norvegicus from Henan and Neimenggu provinces and the other known hantavirus and the vaccine strain. Methods Total RNA was extracted from lung tissues with Trizol reagent. The complete M and S segment sequences of strains NM133 and Q12 were amplified by RT-PCR. The purified DNA fragments were directly subjected to sequencing, and then to sequence analysis and phylogenetic analysis. Results The complete S segment sequences of strains NM133 and Q12 were found to be 1770 nt and 1772 nt in length respectively, with one open reading frame encoding 429 amino acids. The complete M segment sequences of both two strains are 3654 nucleotide in length encoding a protein of 1133 amino acids. The two strains shared a high degree of homology with most of known Seoul virus (SEOV) but quite different from Hantaan virus and other hantaviruses. Furthermore, the nucleoprotein and glycoprotein of the two strains had the congruent structure with the vaccine strain Z37. On the S- and M-phylogenetic trees, both strains (NM133 and Q12) were grouped into the first cluster of SEOV, and were more closely related to the strains, such as: Hb8610, R22, HB55, L99, and K24-e7. Conclusion Both strains (NM133 and Q12) belonged to SEOV, and sharing a high degree of homology and similar secondary structure with strains including the vaccine strains Z37, our data suggested that the present vaccine used in China could effectively prevent HFRS caused by SEOV.
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Objective Complete S and M segments of two Seoul virus (SEOV) strains were obtained to determine their genetic types and characteristics. Methods The complete S and M segments from the isolate Li and lung tissue (sample LF18) were amplified by RT-PCR. Genetic analyses were performed by using DNAStar and PHYLIP program package. Results Their sequences consisted of 1772 nucleotides, and had an open reading frame (ORF, 43 to 1332 nt)encoding a nucleoprotein of 429 amino acids for both two strains. The complete M segment sequences consisted of 3653 nucleotides and had an ORF encoding a GnGc precursor of 1133 amino acids. The GnGc precursor of the two strains had 62 cysteine and 6 N-glycosylation sites. Both two strains shared a high degree of homology with other known SEOV strains including strains L99, Gou3, and vaccine strain Z37, with 87.6% to 99.2% and 83.6% to 97.3% nucleotide identities, respectively. On the S-and M- trees, the two strains LF18 and Li were grouped into the third cluster of SEOV. Conclusion Both LF18 and Li strains belonged to SEOV and shared the congruent genetic characteristics with the vaccine strains Z37. Thus, the bivalent vaccine including the strain Z37 could effectively prevent HFRS which was caused by SEOV in Hebei province.
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Objective Comparing the difference of virulence between the strain CGRn5310 (HTNV) and the strain HR54 (SEOV) isolated both from Rnttus norvegicus. Methods Suckling mice were used to compare the difference of virulence between the two strains. Hantavirus antigens were detected in brain and lung tissues collected from the infected mice. Results Compared with the control group, all infected mice grew slowly. Furthermore, the mice inoculated intracerebrally with either CGRn5310 or HR45 appeared ruffled fur, and reduced activity, followed by neurological symptoms, such as paralyses and convulsions. The half lethal dose (LD_(50)) of CGRn5310 strain was 10~-6.42, whereas the LD_(50) of HR54 strain was 10~-4.51. Hantavirus antigens were identified in brain and lung tissues from the mice infected with the strain CGRn5310 and the strain HR54. Conclusion LD_(50) of the strain CGRn5310 was significantly higher than that of the strain HR54. Our results suggested that the virulence of the spillover hantavirus might only slightly be influenced by the non-reservoir rodents.
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Objective To study the epidemiological features of hantavirus in rodents in Wenzhou,Zhejiang province.Methods Rodents were captured in Wenzhou,where hemorrhagic fever with renal syndrome (HFRS) had been endemic. Hantavirus antigens in the rat lungs were detected by immunofluorescence assay (IFA).Partial S segment (nt 620-999) and partial M segment (nt 2001-2301)sequences were amplified by RT-PCR,and then sequenced.Neighbor-joining method was used to construct for phylogenetic analysis.Results A total of 96 rodents were trapped in the epidemic areas,and 6 hantavirus antigens were identified from these lung samples (6.3%).Partial S and partial M segment sequences were successfully recovered from 5 samples and determined. Phylogenetic analysis of these sequences indicated that all viruses belonged to Seoul virus (SEOV),regardless of the sources (Rattus norvegicus,Rattus tanezumi and Rattus rattoide) that they were derived. However,the clustering pattern in the partial S-tree was different from that in the partial M-tree,suggesting that the re-assortment between SEOVs had occurred.Conclusion All Rattus rats carried SEOV in Wenzhou and the genetic reassortment with SEOV had occurred naturally.
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Objective To study the complete genome sequence of Hantavirus ZT71 strain gene isolated in Zhejiang province and explore its evolution. nethods The total RNA was prepared from ZT71 virus infected cells and the RT-PCR products were cloned into T vector, sequenced and analyzed. Results The L, M and S segments of the strain ZT71 genome were 6530,3651 and 1753 nucleotides in length with a single open reading frame individually encoding 2151,1133 and 429 amino acids. The sequence analysis of nucleotides showed that the homology of L, M and S segments of strain ZT71 between those of other strains of Seoul virus could reach 95.5%-99.7%, 84.1%-99.6% and 88.7%-99.5%, respectively. The analysis of the deduced amino acids showed the similar result. The source of strain ZT71 could be traced from the analysis of the phylogentic trees of nucleotides and amino acids, and it should belong to Seoul type of Hantavirus which was also verified serologically. Conclusion The nucleotide sequences and deduced amino acid sequences of L, M and S segments of strain ZT71 are similar to that of those of Seoul type of Hantavirus. And Hantaan type virus used to be prevalent primarily in Zhejiang province,and it would be an endemic area of mixed type of Hantavirus since the discoveries of the viruses of Soeul type in recent years.
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Background: Hantavirus causes two distinct human diseases: Hemorrhagic Fever with Renal Syndrome (HFRS) and Hantavirus Pulmonary Syndrome (HPS). Hantaviruses are maintained in the rodent reservoir and are transmitted to humans via contaminated excreta or bites. Objectives: This study aims to identificate of Seoul virus in R.novergicus in Hai phong port. Material and method: In this study, we trapped 25 of R. novergicus. Results:The results show four R. novergicus are positive for Seoul virus. Partial M segment sequence was recovered from the lung tissue of R. novergicus trapped in Hai Phong port in 2006. M segment of the Seoul virus of Hai Phong sequenced shown is similar to M segment of the Seoul virus of Hanoi. Conclusion:We discovered the Seoul virus in R.novergicus in Hai Phong port in 2006. The sequence belongs to the Seoul virus genotype and is most closely related to the strain B-1 from Japan and the strain AJ620583 from Indonesia.\r\n', u'\r\n', u'
Subject(s)
Seoul virusABSTRACT
Members of the genus Hantavirus are the etiologic agents of hemorrhagic fever with renal syndrome (HFRS), the diagnosis of which is somewhat difficult because several diseases share similar early clinical presentations such as fever and petechia. In Korea, Hantaan virus and Seoul virus are the causative organisms of HFRS, and the infection caused by Seoul virus is milder than that caused by Hantaan virus. We report a 44-year-old woman, who visited our hospital due to general weakness, fever, myalgia, facial edema and diarrhea. She was diagnosed with HFRS caused by Seoul virus. The antibody against Hantaan virus was positive by an indirect immunofluorescent test and the discrimination between Hantaan and Seoul viruses was done by RT-PCR-RFLP (reverse transcriptionpolymerase chain reaction-restriction fragment length polymorphism) against viral S segment.
Subject(s)
Adult , Female , Humans , Diagnosis , Diarrhea , Discrimination, Psychological , Edema , Fever , Hantaan virus , Orthohantavirus , Hemorrhagic Fever with Renal Syndrome , Korea , Myalgia , Seoul virusABSTRACT
BACKGROUND: Hantaan (HTN) and Seoul (SEO) viruses, harbored by the striped-field mouse(Apodemus agrarius) and the Norway or common rat (Rattus rattus & Rattus norvegicus), respectively, were known to cause hemorrhagic fever with renal syndrome(HFRS) in Korea. We evaluated the seroepidemiologic patterns of hantaviral infections and detect the hantaviral antigens from patients' sera. METHODS: Total 8,102 HFRS patients' sera were collected from 1994 to 1996, and examined by indirect immunofluorescent antibody technique (IFA), hemagglutination inhibition test (HI), IgM emzyme-linked immunosorbent assay (IgM-ELISA) and nested reverse transcription-polymerase chain reaction(RT-PCR). RESULTS: The seropositive rate against hantaviral antigen was 12.0% (973/8102) with the high incidence rate (68.3%) in the period from October to January, and males in the thirties were mostly affected. HTN viral infections were detected 3.5 and 5.2 times higher than SEO viral infections by HI and RT-PCR, respectively, and patients in the fifties were the mostly affected age-group in SEO viral infections, IgM antibodies were detected in the 717 sera of the 905 IFA positive cases (79.2%), and the antigen detection rate of HTN and SEO viruses was 7.7% (56/724). Interestingly, 40 sera (4.4%), showed higher antibody titers against the Puumala (PUU) virus than those against HTN or SEO viruses. CONCLUSION: The results showed HTN and SEO viruses were the main causative agents of HFRS in Korea, and also suggested the possible presence of PUU-related hantaviral infections.
Subject(s)
Animals , Humans , Male , Rats , Antibodies , Fever , Hantaan virus , Orthohantavirus , Hemagglutination Inhibition Tests , Hemorrhagic Fever with Renal Syndrome , Immunoglobulin M , Incidence , Korea , Norway , Puumala virus , Seoul , Seoul virus , Seroepidemiologic StudiesABSTRACT
BACKGROUND: Hantavirus are widely distributed in rodents populations even in geographical areas where hemorrhagic fever with renal syndrome (HFRS) has not been reported. Multiple species of Murid and Arvicolid rodents serve as the natural reservoirs of hantaviruses. Serologic diagnosis using hantaviral antigens indicates that hantaviruses are widely distributed in wild rodents. This study was designed to find the distribution of hantaviruses among wild rodents and small mammals in Korea, 1995-1996. METHODS: Rodents were trapped alive in selected areas. A total of 551 wild rodents from 7 species and 97 small mammals from 4 species were captured in Korea. Serologic evidence for hantavirus infection were tested using five hantavirus antigens by indirect immunofluorescent antibody technique (IFA). Among 162 Apodemus agrarius, 23 Apodemus peninsulae, 8 Clethrionomys regulus, 6 Microtus fortis, 1 Mus musculus, 283 Tamias sibiricus, 68 Sciurus vulgaris, 14 Crocidura laciura, 80 Lepus sinensis, 2 Capereolus capereolus and 1 Nyctereutes procyonoides. RESULTS: 29 A. agrarius, 2 A. peninsulae, 1 C. laciura, 2 C. regulus, 27 T. sibiricus and 7 S. vulgaris were seropositive against Hantaan virus and 7 L. sinensis were IF antibody positive against Seoul virus. Some of Tamias sibiricus were only seropositive against Puumala virus or prospect hill virus. CONCLUSION: This data suggests that new serotypes of hantavirus might distribute among rodents in Korea.