Animal and human antibodies reactive with the outer surface protein A and B of Borrelia burgdorferi are borreliacidal, in vitro, in the presence of complement

Abstract Polyspecific antibodies present in ascitic fluids of mice (pMIAFs) immunized with whole Borrelia burgdorferi cells exerted borreliacidal activity in vitro when tested with complement and homologous antigen but not with heterologous B. hermsii . Similarly, monospecific mouse antibodies obtained by immunizing mice with purified preparations of outer surface protein A and B of B. burgdorferi were borreliacidal. On the contrary, mouse monospecific antibodies raised against the 41-kDa flagellar protein of B. burgdorferi did not kill borreliae in the presence of complement. A complement-mediated, in vitro, borreliacidal activity was observed in human sera from patients with Lyme disease when antibodies against OspA and/or OspB were detectable in sera by the Western blotting technique. The in vitro borreliacidal activity of human sera was evident after 14 h incubation with live B. burgdorferi spirochaetes and complement, whereas antibodies present in mouse immune ascitic fluids killed borreliae after 1 h incubation.     

Immunofluorescence Staining of Group B Coxsackieviruses

Studies were conducted on the sensitivity and specificity of indirect fluorescent-antibody (FA) staining for identification of group B coxsackieviruses. Antisera produced in four different species (monkeys, rabbits, horses, hamsters) and immune ascitic fluids prepared in mice were compared for suitability in FA staining. The horse antisera showed high titers of nonspecific staining, and the rabbit antisera showed relatively low homologous FA titers. Immune reagents from monkeys, hamsters, and mice were used for homologous and heterologous testing against cell cultures infected with the various group B coxsackieviruses. Antisera or immune ascitic fluids produced in these three species showed some heterotypic and nonspecific staining at low dilutions, with the monkey antisera showing the highest heterotypic titers. However, the immune reagents could be diluted to a point where they gave no heterotypic reactivity, but still showed characteristic homotypic staining. Heterotypic staining appeared as diffuse, low-level staining of the cells, whereas homotypic staining revealed characteristic, brightly staining aggregates of viral antigen in the cytoplasm of the infected cells. By using hamster immune sera, appropriately diluted to eliminate heterotypic staining and yet give strong homotypic staining, it was possible to identify correctly 79 (93%) of 85 field strains of group B coxsackieviruses at the first passage level in BS-C-1 cells; the remainder of the strains were identified after two passages in BS-CS-1 cells. No incorrect identifications were made. A limited number of field strains of group B coxsackieviruses were passed into rhesus monkey kidney and human fetal diploid kidney cells, and these were all correctly identified by FA staining, even the strains which failed to produce a cytopathic effect in the human fetal diploid kidney cells. Two human heart and brain tissues from which coxsackievirus type B4 had been isolated failed to show homotypic FA staining in excess of nonspecific or heterotypic staining.     

A comparison of galactosyltransferase activity in mouse ascites lymphoma cells (Ly/Ya), in cultured lymphoma cells, in ascitic fluid and culture medium

Comparative studies were carried out on the galactosyltransferase activity in ascites lymphoma cells isolated from mouse with ascitic lymphoma Ly/Ya, in these cells grown in vitro (24 hrs culture), in ascitic fluid and culture medium. The effect of varying amounts of UDP-galactose on transfer rate of galactose to ovomucoid by the cell enzyme (ascitic and cultured lymphoma cells) and by the soluble enzyme (ascitic fluid and culture medium) was studied. The activity of the enzyme in the cell culture medium was 2.5-fold higher than that in ascitic fluid. The apparent Km values for UDP-galactose of the enzyme from both kinds of cells and from the two fluids was 7.14 x 10(-7) M. At saturating concentrations of donor substrate, V values for the cells and culture medium was 765 pmoles/10(6) cells/h and 180 pmoles/10(6) cells/h for the ascitic fluid.     

In vitro recognition of alloantigens: nature of responding and stimulating cells.

Immunosuppressive and immunostimulatory activity of human cancer ascitic fluids has been examined using the in vitro primary plaque-forming cell response (PFR) to sheep erythrocytes (Mishell-Dutton assay). We have prepared three fractions of ascitic fluid by precipitation with ammonium sulfate. Suppressive activity occurred in the fraction which was insoluble at 50% of saturation but not those fractions which precipitated at 30 or 80% of saturation. The fractions which were precipitated at 30 and 80% were stimulatory in the assay system. Normal human serum also had suppressive activity in the fraction precipitated at 50% of saturation but not as much as was found in ascitic fluid. Serum did not yield any fractions with stimulatory activity.     

Antibody and immune complexes induce tissue factor production by human endothelial cells

Patients with systemic lupus erythematosus (SLE) have an increased incidence of arterial and venous thromboses. The mechanism by which thromboses develop in these patients is unknown. We had previously observed that the sera of patients with SLE contain antibodies and immune complexes that can bind to endothelial cells. Because endothelial cells can synthesize tissue factor, a potent activator of coagulation, we studied the effect of IgG complexes and sera from patients with SLE on the production of tissue factor by these cells. Human umbilical venous endothelial cells incubated with heat-aggregated IgG (HA-IgG) (0.5 to 4.0 mg) elaborate procoagulant activity in a dose-dependent manner. All procoagulant activity was found in the particulate cell fraction, and none was secreted into the medium. Maximum expression of procoagulant activity required 6 to 8 hr, and its production was totally inhibited by the addition of cyclohexamide or actinomycin D. The presence of gel-filtered platelets augmented production of procoagulant activity by endothelial cells stimulated by HA-IgG. Endothelial cell procoagulant activity was not inactivated by diisofluoropropylphosphate, required the presence of Factor VII for its expression, and was neutralized by a specific anti-tissue factor antibody. Endothelial cells incubated with sera from 14 of 16 patients with SLE produced increased amounts of tissue factor compared with 21 normal sera (p less than 0.025). Fractions of two SLE sera containing monomeric IgG, IgA, or IgM, as well as fractions containing IgG complexes, each stimulated endothelial cells to produce more tissue factor than similar fractions prepared from two normal sera. These studies demonstrate that endothelial cells will produce the procoagulant tissue factor after exposure to anti-endothelial cell antibodies or IgG-containing immune complexes. The production of tissue factor by endothelial cells at sites of immune vascular injury may play a role in the development of thromboses in patients with SLE.     

A synthetic corticosteroid,dexamethasone regulates generation of soluble form of interleukin-6 receptor of human lymphocytes,in vitro

In contrast to most of the soluble cytokine receptor antagonists properties, the soluble IL-6 receptor (sIL-6R) occurring in various body fluids of healthy persons and patients with various diseases is an agonist. The enhancing effect is due to its ability to form complex with IL-6 and to bind to gp130 making constitutively IL-6 receptor negative cells responsive for IL-6. The generation as well as the functional role of soluble IL-6 receptor is poorly understood. Earlier, we found that the sIL-6R levels in sera of patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) were higher than those of the control group measured by ELISA sandwich technology. In the present study we detected different levels of sIL-6R in the supernatants of lymphocyte cultures of healthy persons and patients with RA as well as SLE. Moreover, we found, that in vitro dexamethasone treatment stimulated generation of sIL-6R in both healthy persons and in active SLE, while it strongly suppressed production of sIL-6R in both RA groups. At mRNA level, we found that in SLE both the IL-6R mRNA encoding the membrane spanning and alternatively spliced (soluble) variants increased. Surprisingly, the strong decrease of sIL6R protein in RA was not found at mRNA level.     

Use of monoclonal antibody to immunochemically characterize variant-specific surface coat glycoprotein from Trypanosoma rhodesiense

Monoclonal antibodies were employed to study the molecular basis for charge heterogeneity in variant-specific surface coat glycoprotein prepared from clone CP3B4 of the Wellcome strain of Trypanosoma rhodesiense. Thirteen hybridomas secreting monoclonal antibodies specific for CP3B4 were obtained by fusing murine plasmacytoma cells to spleen cells from mice immunized with purified surface coat glycoprotein. The clone population of CP3B4 trypanosomes was shown to be homogeneous by means of immunofluorescent assays using culture supernatants from each of the 13 hybridomas. No cross-reactivity was found with other variant antigenic types of the same serodeme. Ascitic fluids were generated from 4 of the hybridomas and th molecular and epitopic specificities of the fluids or their IgG fractions were determined isoelectrofocusing of immunoprecipitates of radioiodinated glycoprotein antigen followed by autoradiography revealed that all 3 major components of the charge heterogeneous CP3B4 surface-coat glycoprotein were immunoprecipitated by each of the 4 monoclonal IgG fractions. Immunofluorescent staining of live trypanosomes was obtained with only one of the 4 ascitic fluids. The results show that charge heterogeneity does not derive from a heterogeneous population of parasites. Furthermore, the data indicate that there are at least 2 different epitopic specificities exhibited by the monoclonal antibodies tested and that each of the 3 charge heterogeneous components of the surface coat glycoprotein contains these epitopes. Charge heterogeneity of CP3B4 surface coat glycoprotein may be attributed to post-translational modification or to limited proteolysis.     

The production and characteristics of an interleukin-6-dependent hybridoma]

The interleukin-6-(IL-6)-alpha dependent B-cell heterohybridomas were obtained by the fusion of X65.Ag8.653 cells with spleen cells from August rats immunized with lipopolysaccharide E. coli. One of these hybridomas (D6C8) was found to be most dependent on IL-6 for its surviving and growth. Human recombinant IL-1 beta and tumor necrosis factor-alpha could not induce the in vitro growth of this cell line. Presence of elevated level of IL-6 was demonstrated in the sera of patients with rheumatoid arthritis. A specific and sensitive detection of the IL-6 activity in test samples makes it possible to study the presence and role of IL-6 in various immunological disorders.     

Comparison of cellular phenotypes in tumor cyst and ascitic fluid from ovarian serous carcinoma.

Density gradient centrifugation was applied to isolate cell subsets from tumor cyst and ascitic fluid in eight patients with ovarian serous carcinoma. A comparison of cellular composition and immunologic reactivity of cells from the cysts and from ascitic fluid in each patient was performed. Some differences in density profiles were found, but in each case the consistency of morphologic cell forms in the primary tumor and ascites was documented. Immunophenotypic analyses of isolated cellular fractions using polyclonal and monoclonal antibodies against ovarian carcinoma-associated antigens showed significant immunologic intratumoral heterogeneity. However, there was a similarity of antigen expression in cells from the primary tumors and ascitic fluids. Our study indicated that morphologic and antigenic characterization of a given tumor could be determined in a single representative sample of ascitic fluid.     

Production of B cell-stimulating factors by B cells in patients with systemic lupus erythematosus

The production of B cell-stimulating factors (BSF) by B cells in patients with systemic lupus erythematosus (SLE) was studied in vitro. B cells from SLE patients markedly proliferated and differentiated into Ig-producing cells by in vitro culture without any stimulation. The culture supernatant of these B cells contained BSF activity that stimulated Staphylococcus aureus Cowan I-treated normal B cells to proliferate and differentiate into Ig-producing cells. By a Percoll gradient density centrifugation, BSF-producing cells were enriched in the higher density fraction, but were reduced in the lower density fraction. The BSF also stimulated the proliferation and the differentiation of SLE B cells. By a Percoll gradient density centrifugation, SLE B cells responsive to the BSF were enriched in the higher density fraction, but were reduced in the lower density fraction. The Mr of the BSF was estimated as about 18,000 Da by Sephacryl S-200 column chromatography. The BSF fraction did not possess IL-2 and IFN activity, but possessed IL-1 activity, which stimulated murine thymocyte proliferative responses. The BSF activity was partially, but not completely, absorbed by an anti-IL-1 alpha antibody. Furthermore, the BSF possessed IL-4 activity, which induced not only the proliferative responses of normal B cells stimulated with B cell mitogens, but also the expression of low affinity Fc epsilon R/CD23 on normal B cells. The BSF also possessed IL-6 activity, which induced the proliferative responses of IL-6-dependent hybridoma cells, MH-60 BSF2. Moreover, human rIL-1, rIL-4, and rIL-6 stimulated SLE B cells. These results suggest that SLE B cells spontaneously produce the BSF such as IL-1 alpha, IL-4, and IL-6 and express their receptors on their surface, and the interaction between the BSF and their receptors stimulates SLE B cells to spontaneously proliferate and differentiate into Ig-producing cells as an autocrine mechanism.     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.     

The occurrence of the feline A blood group antigen on lymphocytes

Sera from 300 cats were tested for the presence of anti-lymphocytic antibodies. One hundred and nineteen sera showed some activity with the majority (79) reacting only with lymphocytes from blood group A cats. Absorption of two such sera with A, AB and B erythrocytes and absorption of AB system reagents with lymphocytes from A and B blood group cats demonstrated that the A antigen is expressed on both erythrocytes and lymphocytes. Blood group and lymphocyte typing tests of foetuses indicated that the A antigen is present on these tissues as early as 46 days gestation. The erythrocytic B antigen could not be demonstrated on lymphocytes although a single antiserum, which reacted against lymphocytes from group B cats, was found. Several sera containing anti-lymphocytic antibodies which were not related to the AB type were also detected.     

Reversal of the Progression of Fatal Coronavirus Infection in Cats by a Broad-Spectrum Coronavirus Protease Inhibitor

Coronaviruses infect animals and humans causing a wide range of diseases. The diversity of coronaviruses in many mammalian species is contributed by relatively high mutation and recombination rates during replication. This dynamic nature of coronaviruses may facilitate cross-species transmission and shifts in tissue or cell tropism in a host, resulting in substantial change in virulence. Feline enteric coronavirus (FECV) causes inapparent or mild enteritis in cats, but a highly fatal disease, called feline infectious peritonitis (FIP), can arise through mutation of FECV to FIP virus (FIPV). The pathogenesis of FIP is intimately associated with immune responses and involves depletion of T cells, features shared by some other coronaviruses like Severe Acute Respiratory Syndrome Coronavirus. The increasing risks of highly virulent coronavirus infections in humans or animals call for effective antiviral drugs, but no such measures are yet available. Previously, we have reported the inhibitors that target 3C-like protease (3CLpro) with broad-spectrum activity against important human and animal coronaviruses. Here, we evaluated the therapeutic efficacy of our 3CLpro inhibitor in laboratory cats with FIP. Experimental FIP is 100% fatal once certain clinical and laboratory signs become apparent. We found that antiviral treatment led to full recovery of cats when treatment was started at a stage of disease that would be otherwise fatal if left untreated. Antiviral treatment was associated with a rapid improvement in fever, ascites, lymphopenia and gross signs of illness and cats returned to normal health within 20 days or less of treatment. Significant reduction in viral titers was also observed in cats. These results indicate that continuous virus replication is required for progression of immune-mediated inflammatory disease of FIP. These findings may provide important insights into devising therapeutic strategies and selection of antiviral compounds for further development for important coronaviruses in animals and humans.     

Transforming growth factor-beta does not alter interleukin-1 expression in cultured human macrophages

Transforming growth factor-beta (TGF beta) is a growth modulator that stimulates the growth of fibroblastic cells but inhibits the growth of cells of epithelial origin. TGF beta also influences the production of extracellular matrix proteins, and of proteases and the type 1 plasminogen activator inhibitor (PAI-1) by cultured cells. TGF beta appears also to have various immunoregulatory effects, suppressing both T- and B-cell activities. It has been proposed that it might increase the expression of interleukin-1 (IL-1) mRNA in cultured human monocytes, thus potentiating immune functions. To analyze the role of TGF beta in IL-1 production we have now quantitated the effect of this factor on the production of biologically active IL-1 as well as IL-1 beta mRNA expression. The effect of TGF beta on IL-1 production optimally activated with bacterial lipopolysaccharide (LPS) was also studied. It was found that IL-1 activity and mRNA levels were rapidly elevated by LPS but not by TGF beta. Culture fluids from monocytes treated with TGF beta alone or with TGF beta plus LPS inhibited the proliferation of the test thymocytes. After gel filtration, the media from TGF beta-treated cultures showed no activity in the molecular weight area of IL-1 (approx. 15 kD), while the supernatants from TGF beta plus LPS-induced cells contained IL-1 activity in these fractions, the magnitude of which was, however, at the same level as in the culture fluids derived from cells stimulated with LPS alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in cytokine production and morphology of murine lymphoma NK/Ly cells in course of tumor development

The main goal of this study was to evaluate if specific cytokine expression in the NK/Ly lymphoma cells might be involved in development of intoxication in the tumor-bearing animals. RT-PCR analysis was used to study an expression of mRNA coding for IL-1α, IL-6, TNF-α, TNF-β and VEGF. ELISA was used to evaluate IL-6 and IFN-γ concentration in the ascitic fluid. Cytomorphological investigation of tumor cells was done after standard Romanovsky-Giemsa staining, and chromatin staining was performed with hematoxyline and neutral red. Lactate dehydrogenase and acid phosphatase release from tumor cells was estimated. It was revealed that the level of mRNA coding for VEGF and IL-6 was significant in the lymphoma cells. The level of VEGF mRNA was initially high and did not change during tumor progression, while the level of expression of IL6 mRNA was low at the initial stages of tumor growth and markedly increased (up to 5-fold) at the terminal stages. The obtained data on IL-6 mRNA expression were confirmed by ELISA, which showed more than 6-fold increase (from 90 to 570 pg/ml) in the IL-6 concentration in the ascitic fluid at late stages of NK/Ly tumor development. On the contrary to IL-6, concentration of IFN-γ in the ascitic fluid was very high at early stages of tumor development (1,000 pg/ml) and it markedly decreased (up to 30-fold, 30 pg/ml) at the terminal stages of tumor development. The high levels of IL-6 mRNA in tumor cells and IL-6 content in extracellular medium correlated with cell deterioration, as revealed by cytomorphologic study and the release of intracellular enzymes into extracellular medium. We suggest that an enhanced production and release of IL-6 by lymphoma cells can cause intoxication and exhaustion of the organism observed at terminal stages of tumor growth.     

Identification of IL-6 as a T cell-derived factor that enhances the proliferative response of thymocytes to IL-4 and phorbol myristate acetate

Thymocytes undergo a vigorous proliferative response when stimulated with a combination of IL-4 and PMA. We have found that conA-induced supernatants from a number of Th cell clones could enhance the level of IL-4/PMA-induced proliferation of unseparated thymocytes 0.5- to 2-fold and of peanut agglutinin-positive thymocytes 2- to 10-fold. These supernatants did not contain IL-2 or IFN-gamma, and the enhancing activity could be chromatographically separated from IL-3, -4, -5, and granulocyte/macrophage CSF. The possibility that the thymocyte enhancement factor contained in these supernatants was IL-6 was suggested when murine rIL-6 was found to have similar activity. Further evidence for the identity of these two factors was obtained when an IL-6 assay, based on plasmacytoma growth, was used to test column fractions showing thymocyte enhancement. All fractions active in the thymocyte enhancement assay also had activity in the plasmacytoma growth assay. These observations suggest that the thymocyte-stimulating activity present in the T cell supernatants was due to IL-6.     

Natural killer cell cytotoxic potential of patients with ovarian carcinoma and its modulation with virus-modified tumor cell extract

Summary We have demonstrated that cancer patients with ovarian carcinoma display deficient peripheral blood NK-cell cytotoxic potential against the K-562 target cell line. Furthermore, no NK-cell activity against the same tumor was detected in ascitic fluids of these patients. The inferior peripheral blood NK-cell cytotoxicity of ovarian carcinoma patients was significantly augmented after ID inoculation with virus-modified tumor cell extract. Similarly, NK-cell activity in the ascitic fluids was dramatically increased after IP in vivo therapy with the same tumor extract preparation. Interestingly, in some of the cancer patients the augmentation of NK-cell activity in ascitic fluids after IP injection of virus-modified tumor cells extract was associated with a clinical response of the patients, as demonstrated by regression of ascitic tumors. These studies indicate, first, that virus-modified tumor extract displays immunopotentiating activity, as reflected by its marked NK cell-augmenting potential, and secondly, that regional activation of NK cells could underlie the mechanism of regression of ascitic tumors.     

Regulated expression of galectin-1 during T-cell activation involves Lck and Fyn kinases and signaling through MEK1/ERK,p38 MAP kinase and p70S6 kinase

It has been shown that staphylococcal enterotoxin A (SEA) acts through human peripheral blood mononuclear cells (PBMC) to stimulate synthesis or release of pyrogenic cytokines. Nuclear factor-kappa B (NF-kappaB) is thought to play an important role in inflammatory responses through the regulation of genes encoding pro-inflammatory cytokines. The purpose of the present study was to determine whether the NF-kappaB mechanisms in human PBMC are involved in SEA-induced fever. Western blot evaluation revealed SEA was able to induce nuclear translocation of NF-kappaB from cytosol to nucleus in PBMC, which could be abolished by a NF-kappaB inhibitor such as pyrrolidine dithiocarbamate (PDTC), sodium pyrithione (Pyri), N-acetyl-L-cysteine (NAC), or curcumin (Cur). Electrophoretic mobility shift assay also showed that the NF-kappaB DNA-binding activity was increased in the SEA-treated PBMC. Again, the SEA-induced increased NF-kappaB binding activity was significantly attenuated by either PDTC, Pyri, NAC or Cur. The pyrogenic responses to supernatant fluids obtained from human PBMC stimulated with SEA were associated with increased levels of interleukin 1-beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha) in the supernatant fluids. Both the fever and the increased levels of IL-1beta, IL-6, and TNF-alpha in supernatant fluids obtained from the SEA-stimulated PBMC were decreased by incubating SEA-PBMC with either PDTC, Pyri, NAC, or Cur. Furthermore, the fever induced by systemic or central administration of SEA in rabbits were attenuated by pre-treatment with an systemic or central dose of either PDTC, Pyri, NAC, or Cur. The data indicate that inhibition of NF-kappaB prevents SEA-induced fever.     

Secreted MUC1 mucins lacking their cytoplasmic part and carrying sialyl-Lewis a and x epitopes from a tumor cell line and sera of colon carcinoma patients can inhibit HL-60 leukocyte adhesion to E-selectin-expressing endothelial cells

A secreted MUC1 mucin from the spent medium of the colon carcinoma cell line COLO 205 carrying sialyl-Lewis a and x epitopes (H-CanAg) was purified by trichloroacetic acid precipitation and Superose 6 gel filtration. The purified H-CanAg inhibited adhesion of the leukocyte cell line HL-60 to E-selectin transfected COS-1 cells or interleukin-1β (IL-1β)-activated human umbilical vein endothelial cells. Sera from two patients with advanced colon carcinoma containing high concentrations of sialyl-Lewis a and x activity inhibited HL-60 cell adhesion to E-selectin-expressing COS-1 cells and IL-1β-activated endothelial cells. After affinity column absorption of the sialyl-Lewis a activity, the sera also lost most of their sialyl-Lewis x activity and at the same time their adhesion inhibitory effect. A large part of the sialyl-Lewis a/x activity in the two patients was found in fractions containing mucins having a MUC1 apoprotein, as shown by its size, and reactivity with the two anti-MUC1 apoprotein monoclonal antibodies, Ma552 and HMFG-2. The cell-adhesion inhibitory effect of the purified sialyl-Lewis a-carrying MUC1 mucin fraction from the sera of the two patients was stronger than that of smaller sized sialyl-Lewis a-carrying mucin-type glycoproteins also found in the patient sera. The MUC1 mucin fraction secreted by the COLO 205 cells and from the two sera were all shown to lack their C-terminal portion, in contrast to the MUC1 mucin from cells. It is hypothesized that sialyl-Lewis a- and/or x-containing mucins, especially MUC1, secreted by tumors can interact with E-selectin on endothelial cells and thus inhibit leukocyte adhesion. © 1996 Wiley-Liss, Inc.     

我国新分离虫媒病毒的初步鉴定

1990－1994年,从新疆地区的蚊、蜱和病人血清分离了多株病毒,为了明确这些病毒的分类地位,对其中的20株病毒进行了组织培养细胞感染实验和血清学检验,对部分毒株做了动物接种实验和理化性质鉴定。结果显示：20株病毒均可使BHK－21细胞病变（1－3天）,主要表现为细胞圆宿、聚集,融合,破碎,脱落等;致Vero细胞病变为2－4天;15株病毒致C6／36细胞病变（2－4天）,5株病毒对C6／36细胞连续观察7天未见细胞病变。11株病毒对乳鼠2－4天致死,对成年鼠2－5天致死。选取6株病毒进行理化性质鉴定,4株病毒（90260、91002、91004和91028）对5－氟脱氧尿苷耐受,对乙醚和酸敏感,提示为有膜RNA病毒;一株病毒（90265）对5－氟脱氧尿苷、乙醚和酸均敏感,提示为有膜DNA病毒;另一株病毒（9059）对5－氟脱氧尿苷耐受,对乙醚和酸也耐受,提示可能为无膜RNA肠道病毒。20株病毒中,17株病毒与甲病毒、乙型脑炎病毒和布尼亚病毒的特异性免疫腹水不反应,提示这些病毒中可能不存在甲病毒、黄病毒和布尼亚病毒;3株病毒（90260、91002和91004）只与甲病毒的特异性免疫腹水反应,与乙型脑炎病毒和布尼亚病毒的不反应,提示这三株病毒为甲病毒。