The purpose of this study is to delineate the histopathologic findings of the spleen after Hantaan viral inoculation, which is the largest lymphoid organ in rats, and to identify the viral location by anti-Hantaan virus (HTNV) monoclonal antibody. All the sixty one suckling rats of less than twenty four hours of age were used. Except twenty one rats of control group, twenty-five rats inoculated intracerebrally for the early change and fifteen suckling rats inoculated intramuscularly for the late change were uniformly susceptible to lethal infection with the ROK 84-105-1 strain of seed HTNV. The characteristic histopathologic findings were; appearance of macrophages below the splenic capsule on the 3rd day, small lymphocytes around the periarteriolar sheath on the 5th day increasing in numbers on the 7th day, and a markedly expanded marginal zone with some immunoblasts and plasma cells as well as decreased extramedullary hematopoiesis on the 9th and 14th days. Time of onset of histopathologic changes in spleen thickness, appearance of medium and large lymphocytes and degree of extramedullary hematopoiesis were influenced by inoculation route, whereas expansion of the marginal zone was affected by postnatal age.
Animals ; Animals, Suckling ; Antigens, Viral/analysis ; Hantavirus/immunology ; Hematopoiesis ; Hemorrhagic Fever with Renal Syndrome/*pathology ; Rats ; Rats, Inbred Strains ; Spleen/*pathology

BACKGROUNDTo express and purify human alpha3-integrin to serve as the antigen to prepare its antibody and to separate the Vero cell clones without expression of alpha3-integrin.

METHODSThe human alpha3-integrin gene was amplified by using RT-PCR, then subcloned into a pQE30 expression vector and expressed in E. coli. The gene expression was confirmed by Western blot assay. Rabbit was inoculated with purified antigen to stimulate the antibody generation. The target Vero cells were separated by negative selection using antibody plus complement mediated cytolysis. The separated cell clones were confirmed by immunofluorescence and Western blot assay.

RESULTSThe alpha3- integrin gene was cloned and expressed effetively, Western blot assay revealed that the expressed protein held good immune reactivity. High titer antibody was generated. However the expression of alpha3-integrin was not detected on Vero, VeroE6, Hep-2, 2BS and 293 cells.

CONCLUSIONSThe results of the study suggested that hantavirus has other receptors on Vero cells beside alpha 3-integrin.

Animals ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Hantavirus ; Integrin beta3 ; biosynthesis ; genetics ; immunology ; Rabbits ; Receptors, Virus ; Vero Cells ; metabolism

OBJECTIVETo evaluation the effect of different mucosal vaccination pathway on hantavirus with the recombinant E. coli heat-labile enterotoxin B subunit (rLTB) as adjuvant.

METHODSThe rLTB was expressed and purified. Take the inactivated hantavirus strain 84Fli as vaccine, and immunized C57 BL/6 mice through intranasal, oral and vaginal respectively. Specific IgG and sectory IgA were detected by ELISA in serum, and vaginal washing samples respectively.

RESULTSThe rLTB was efficiently expressed under the induction of lactose, identified by western blotting and GM-1 binding experiment. The vaccination through intranasal, oral and vaginal, can induce IgG and sectory IgA response.

CONCLUSIONInactivated hantavirus can produce mucosal immune response with rLTB as adjuvants through intranasal, oral and vaginal vaccination respectively.

Adjuvants, Immunologic ; administration & dosage ; genetics ; Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; administration & dosage ; genetics ; immunology ; Enterotoxins ; administration & dosage ; genetics ; immunology ; Escherichia coli Proteins ; administration & dosage ; genetics ; immunology ; Female ; Hantavirus ; genetics ; immunology ; Hantavirus Infections ; immunology ; virology ; Humans ; Immunity, Mucosal ; Immunoglobulin A ; blood ; Immunoglobulin G ; blood ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Viral Vaccines ; administration & dosage ; genetics ; immunology

OBJECTIVETo develop a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) for the detection of HTNV IgM antibody.

METHODSBlack solid 96 well microplate was coated with anti-human IgM-microantibody, HRP labeled HTNV recombinant nucleotide antigen was used as detection antigen, luminol-H2O2 was used as substrate, a CLEIA was established for the detection of HFRS patient serum IgM antibody and comparison of detection sensitivity, specificity, and stability were made between CLEIA and MacELISA.

RESULTSCorrelate coefficient of CLEIA with MacELISA is 0.97; detection sensitivity of CLEIA is 100 percent while that of MacELISA is 92.1 percent; detection specificity of CLEIA and MacELISA are both 100 percent; coefficient of variance for intra-assay and inter-assay of CLEIA are both less than 15 percent, which are comparative with MacELISA.

CONCLUSIONThe established method of CLEIA is a sensitive, selective, and stable method; it is suitable for the early detection of HFRS patient serum IgM antibody.

Antibodies, Viral ; blood ; Antibody Specificity ; Enzyme-Linked Immunosorbent Assay ; methods ; Hantavirus ; immunology ; Hemorrhagic Fever with Renal Syndrome ; immunology ; Humans ; Immunoglobulin M ; blood ; Luminescent Measurements ; methods

BACKGROUNDTo observe the features of serum specific IgA, IgG, IgM antibodies in the acute phase of hemorrhagic fever renal syndrome (HFRS).

METHODSThe nucleocapsid (NP) protein and glycoproteins (GP) of Hantavirus were expressed by recombinant baculovirus, and used as ELISA antigens to test 61 serial sera of 14 acute phase HFRS patients.

RESULTSSeoul like virus RNA were detected from 11 of 14 patients. An early and strong IgA, IgG and IgM antibody response to recombinant NP (rNP) was observed in almost all HFRS cases. The titers of antibody to rNP was apparently higher than that to Rgp. In the early stage, titer of IgG antibody elevated most drastically among all the three classes of antibodies to rNP, followed by IgM and IgA antibody responses. The elevation trend of IgM and IgA antibodies to rNP stayed nearly at the same level, but the IgA titers to rNP were apparently higher than that of IgM. Among the antibodies to rGP, IgA changed distinctly greater than IgG. The elevation trend of IgM could be found during first week after the onset, and the titers dropped gradually after the second week. IgM antibodies of one case who was viral RNA positive were not detected at early stage, but IgA titers were high. The only severe case of the 14 patients kept the lower IgA, IgG and IgM during the whole acute phase.

CONCLUSIONHFRS patients kept an early and strong humoral response to NP and GPs in acute phase of HFRS.IgA could be used together with IgM to improve the diagnostic accuracy.

Acute Disease ; Adult ; Antibodies, Viral ; blood ; Capsid Proteins ; genetics ; immunology ; Female ; Hantavirus ; genetics ; immunology ; Hemorrhagic Fever with Renal Syndrome ; immunology ; virology ; Humans ; Immunoglobulin Isotypes ; blood ; Male ; Viral Core Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

BACKGROUNDThe heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus.

METHODSBALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleocapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the humoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization.

RESULTSAntigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1:50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 co-injection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation.

CONCLUSIONHumoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by co-inoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

Animals ; Cytokines ; biosynthesis ; Genetic Therapy ; Hantavirus ; immunology ; Immunoglobulin G ; blood ; Immunophenotyping ; Interleukin-12 ; genetics ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Nucleocapsid ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

Hemorrhagic fever with renal syndrome in Korea (Korean hemorrhagic fever) is an acute viral disease characterized by fever, hemorrhage and renal failure. In Korean patients, the disease manifests more distinctive bleeding tendencies than those of hemorrhagic fever with renal syndrome found in western countries. To investigate the nature and role of the coagulation, fibrinolysis, kinin and immune system in the pathogenesis of such a hemorrhagic manifestation, alterations of these systems were assessed from the early phase of the disease. Decreased platelet count and shortened platelet survival were observed with giant platelets in the peripheral blood. The marked prolongations of bleeding time, prothrombin time and partial thromboplastin time were noticed with the decreased plasma activities of coagulation factors II, V, VIII, IX and X. Shortened half life of fibrinogen, increased fibrinogen-fibrin degradation product, with decreased plasma levels and activities of plasminogen, alpha 2-plasmin inhibitor and antithrombin III were found. On thrombelastogram, the existence of procoagulant activity was confirmed, and prolonged reaction time and clot formation time with decreased maximum amplitude were observed. The appearance of circulating immune complexes was found along with decreased C3 and normal C4 in the serum. Significant decrease of serum C3 was evident in the patients with disseminated intravascular coagulation. These findings of coagulopathy were normalized within ten days of the illness in most cases. Therefore, it can be concluded that disseminated intravascular coagulation and thrombocytopenia in the early phase, and azotemia developing later might play an important role in the pathogenesis of bleeding tendency in Korean hemorrhagic fever.
Acute Kidney Injury/*complications ; Blood Coagulation Disorders/*etiology ; Bone Marrow/pathology ; Complement System Proteins/metabolism ; *Hantavirus ; Humans ; Korea ; Virus Diseases/*complications/ethnology/immunology

OBJECTIVEIn order to find out the factors related to hemorrhagic fever with renal syndrome (HFRS) infection, and to evaluate the probability of ecdemic hantaviruses (HV) infection in rodents in Beijing areas.

METHODSRodents were collected in a large-scale railway station and a produce market with 'trap nights' method from April to May, 2004. The IgG reacting sera to HV antigen were detected using ELISA. The partial M and S segment of HV from captured rodent lung samples were amplified with RT-PCR. The PCR products were purified and sequenced. BLAST program was then used to perform on nucleotide pairwise alignment with all available sequence in GenBank. The alignment of the multiply nucleotide and the deduced amino acid sequences, together with phylogenetic analysis were completed with DNASTAR software.

RESULTSThe average population density was 3.49% (24/690). The overall seroprevalence of HV infection was 8.3% (2/24). RT-PCR positive rates were 8.3% (2/24). The nucleotide sequences of 356 bp region (1958 - 2313) of M segment obtained from 2 samples were all identified to Seoul virus (SEOV), with 7.6% heterogeneity. The dc501 strain from railway station was closely related to SD227 and Hebei4 from Shandong and Hebei provinces respectively. BjFT01 strain from the farm product market had more special nucleotide transitional mutations than other known SEOV from Beijing in GenBank. This strain, together with known HN71 from Hainan province, K24-E7 from Zhejiang province, L99 from Jiangxi province and R22 from Henan province, represented a monophylogentic linkage.

CONCLUSIONThe higher HV prevalence of rodents in transportation center was the potential and important risk for HFRS epidemic in Beijing. The increasing prevalence of M. musculus should call for attention. It was possible that SEOV in Beijing was imported by infected rodents through vehicles from other provinces.

Animals ; Antigens, Viral ; immunology ; China ; epidemiology ; Enzyme-Linked Immunosorbent Assay ; Hantavirus ; classification ; genetics ; isolation & purification ; Hantavirus Infections ; epidemiology ; immunology ; Hemorrhagic Fever with Renal Syndrome ; epidemiology ; Immunoglobulin G ; blood ; Lung ; virology ; Phylogeny ; Reverse Transcriptase Polymerase Chain Reaction ; Rodent Diseases ; epidemiology ; virology ; Rodentia ; Seroepidemiologic Studies

BACKGROUNDTo observe cytopathogenic effect of Hantaan virus (HV) on cultured human bone marrow cells.

METHODSLight and transmission electron microscopy and direct immunofluorescent technique were applied to study cellular structure especially ultrastructural changes of bone marrow cells from patients with Hantaan virus infection. Bone marrow cells of one healthy volunteer were also studied as control.

RESULTSThe antigen of HV was found in bone marrow cells of 20 of 27 HFRS patients by the aid of direct immunofluorescent technique. It was found that the granulocytes had the highest percentage of HV antigen positive cells (76%), followed by monocytes (65%), lymphocytes (40%), megakaryocytes (20%) and the lowest was found in erythrocytes (3.7%). The injury of cell membrane after infection with HV was significantly more severe than that in the control group under the light and electron microscopy.

CONCLUSIONThis study demonstrated that HV could attack human bone marrow cells and cause cytopathogenic effect on them.

Adult ; Aged ; Antigens, Viral ; analysis ; Bone Marrow Cells ; ultrastructure ; virology ; Female ; Fluorescent Antibody Technique, Direct ; Hantavirus ; immunology ; pathogenicity ; Hemorrhagic Fever with Renal Syndrome ; pathology ; virology ; Humans ; Male ; Microscopy, Electron, Scanning ; Microscopy, Fluorescence ; Middle Aged