Cases of hemorrhagic fever with renal syndrome in Amur Province associated with the rat serotype of the virus

To carry out serodiagnosis and to determine the serotype of the virus causing hemorrhagic fever with the renal syndrome (HFRS), paired sera obtained from 28 HFRS patients, 42 persons with a history of HFRS (1-17 years after convalescence), and 268 serum samples from healthy persons residing at the areas with the natural foci of this infection have been studied by indirect immunofluorescence techniques at the territory of the Amur Province. This study has demonstrated for the first time that, alongside the diseases caused by HFRS virus of serotype Apodemus, HFRS viruses of serotype Rattus and an unidentified serotype serve as the source of infection for the population of the Amur region. The leading role of serotype Rattus in HFRS has been confirmed by the detection of antibodies mainly to this serotype in serum samples taken from convalescents after HFRS and from the healthy population of the areas with the foci of HFRS virus infection.     

Dobrava, but not Saaremaa, hantavirus is lethal and induces nitric oxide production in suckling mice

Hantaviruses are the causative agents of HFRS and HCPS (hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome), two severe, and often fatal human diseases. Mortality from HFRS varies between hantaviruses; Hantaan and Dobrava show the highest, Seoul intermediate, and Puumala low mortality. Saaremaa, genetically closely related to Dobrava, is also known to induce HFRS, with low or no mortality. In this study, mice were inoculated with Dobrava and Saaremaa viruses to test for infectibility, lethality, viremia, nitric oxide production and antibody responses. Out of suckling mice intracerebrally inoculated with 50, 500 and 5,000 focus-forming units of Dobrava virus, respectively, 1/8, 2/8 and 7/8 died within 18-26 days. In all but one of the lethally infected mice high levels of replicating virus were detected, and most were positive for neutralizing antibodies and showed elevated levels of nitric oxide production. All suckling mice intracerebrally inoculated with 50, 500, or 5,000 focus-forming units of Saaremaa virus survived and all seroconverted. Clearly lower viral titers were observed for the Saaremaa virus-inoculated mice, also when sacrificed at day 18 after infection, compared to those in mice that died following Dobrava virus infection. Dobrava, Saaremaa, Puumala and Hantaan virus infections of adult mice were asymptomatic, and the anti-nucleocapsid protein IgG2a/IgG1-titer ratio was higher in mice inoculated with Dobrava virus than in those inoculated with Saaremaa virus. Elevated nitric oxide production was not detected in asymptomatically infected mice, and iNOS-/- mice, like normal mice, cleared viremia. In conclusion, we show that Dobrava virus and Saaremaa virus induce distinct differences in terms of survival, viremia, nitric oxide production and antibody responses in mice.     

Control of laboratory acquired hemorrhagic fever with renal syndrome (HFRS) in Japan

By the end of 1985, 126 human cases of laboratory acquired hemorrhagic fever with renal syndrome (HFRS) were recorded in Japan. Seroepidemiological studies revealed that laboratory rats exhibited high IFA titers against Hantaan or related viruses at locations where HFRS patients occurred. Laboratory researchers contracted HFRS more frequently than laboratory animal technicians or caretakers, although a laboratory animal caretaker died of the disease. Inhalation of HFRS-virus contaminated air in an animal facility is the likely cause of infection with this virus. Wound infection during animal experiments may be another important route of infection. Infection of laboratory rats can occur by transferring animals from contaminated to other animal facilities. Tissue fragments or cells of transplantable animal tumors are a potential source of spreading the HFRS virus. Eradication of HFRS virus from a contaminated animal facility can be achieved best by elimination of all animals in the room, especially when human HFRS is associated with an infected colony. In some cases, when IFA titers of the sera of the rats tested were low, infection apparently disappeared without instituting any particular control measures other than ordinary procedures for care and management of laboratory animals. HFRS viruses have not yet been eradicated from all animal facilities in Japan. Therefore, serological monitoring of laboratory rats continues.     

东北三省6株肾综合征出血热病毒生物学性状的研究

肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)是我国重点防治的急性传染病,对于流行于不同地区的汉坦病毒(hanatavirus)进行分离和系统地鉴定,可以为HFRS的防治提供科学依据,特别是对流行株血清型及基因型的确定,是研制疫苗和确定防治重点及对策的重要前提,也为研究我国汉坦病毒的遗传和进化特征奠定了基础。研究对6株流行于东北三省的汉坦病毒进行了全面鉴定,其中H8205、H8207株为人源性汉坦病毒,其余均为鼠源性汉坦病毒。研究表明,6株病毒具有病毒型别明确,抗原性较为广谱,免疫原性好,在Vero细胞上适应能力强及病毒滴度高,以及来源及传代历史清楚等特点,适合作为双价HFRS纯化灭活疫苗候选毒株,并且可建立起完善和合格的生产用毒种库,为我国生产以Vero细胞为基质的双价肾综合征出血热纯化灭活疫苗奠定了坚实的基础。     

应用微量细胞病变中和试验法(MCPENT)对我国EHF病毒株和病人血清进行血清分型

本文应用微量细胞病变中和试验(MCPENT)对我国22株不同来源的流行性出血热病毒(EHFV)进行血清分型研究结果除一株具有广谱抗原性外,所有毒株均可准确地被分为血清1型和血清2型,其分型结果与用空斑减少中和试验(PRNT)完全一致。此外,MCPENT法还能对不同流行区EHF病人血清进行分型诊断。此法简便、经济、准确可靠,便于推广应用。     

The multimerization of hantavirus nucleocapsid protein depends on type-specific epitopes

Multimerization of the Hantaan virus nucleocapsid protein (NP) in Hantaan virus-infected Vero E6 cells was observed in a competitive enzyme-linked immunosorbent assay (ELISA). Recombinant and truncated NPs of Hantaan, Seoul, and Dobrava viruses lacking the N-terminal 49 amino acids were also detected as multimers. Although truncated NPs of Hantaan virus lacking the N-terminal 154 amino acids existed as a monomer, those of Seoul and Dobrava formed multimers. The multimerized truncated NP antigens of Seoul and Dobrava viruses could detect serotype-specific antibodies, whereas the monomeric truncated NP antigen of Hantaan virus lacking the N-terminal 154 amino acids could not, suggesting that a hantavirus serotype-specific epitope on the NP results in multimerization. The NP-NP interaction was also detected by using a yeast two-hybrid assay. Two regions, amino acids 100 to 125 (region 1) and amino acids 404 to 429 (region 2), were essential for the NP-NP interaction in yeast. The NP of Seoul virus in which the tryptophan at amino acid number 119 was replaced by alanine (W119A mutation) did not multimerize in the yeast two-hybrid assay, indicating that tryptophan 119 in region 1 is important for the NP-NP interaction in yeast. However, W119A mutants expressed in mammalian cells were detected as the multimer by using competitive ELISA. Similarly, the truncated NP of Seoul virus expressing amino acids 155 to 429 showed a homologous interaction in a competitive ELISA but not in the yeast two-hybrid assay, indicating that the C-terminal region is important for the multimerization detected by competitive ELISA. Combined, the results indicate that several steps and regions are involved in multimerization of hantavirus NP.     

Monitoring Antigenic Variations of Enterovirus 71: Implications for Virus Surveillance and Vaccine Development

Enterovirus 71 (EV71) causes life-threatening epidemics in Asia and can be phylogenetically classified into three major genogroups (A∼C) including 11 genotypes (A, B1∼B5, and C1∼C5). Recently, EV71 epidemics occurred cyclically in Taiwan with different genotypes. In recent years, human studies using post-infection sera obtained from children have detected antigenic variations among different EV71 strains. Therefore, surveillance of enterovirus 71 should include phylogenetic and antigenic analysis. Due to limitation of sera available from children with EV71 primary infection, suitable animal models should be developed to generate a panel of antisera for monitoring EV71 antigenic variations. Twelve reference strains representing the 11 EV71 genotypes were grown in rhabdomyosarcoma cells. Infectious EV71 particles were purified and collected to immunize rabbits. The rabbit antisera were then employed to measure neutralizing antibody titers against the 12 reference strains and 5 recent strains. Rabbits immunized with genogroup B and C viruses consistently have a lower neutralizing antibody titers against genogroup A (≧8-fold difference) and antigenic variations between genogroup B and C viruses can be detected but did not have a clear pattern, which are consistent with previous human studies. Comparison between human and rabbit neutralizing antibody profiles, the results showed that ≧8-fold difference in rabbit cross-reactive antibody ratios could be used to screen EV71 isolates for identifying potential antigenic variants. In conclusion, a rabbit model was developed to monitor antigenic variations of EV71, which are critical to select vaccine strains and predict epidemics.