Isolation of Inoue-Melnick virus from cerebrospinal fluid of patients with epidemic neuropathy in Cuba.

BACKGROUND: A mysterious disease spread over Cuba from 1991 to 1993, the cause of which has not been fully established. Major symptoms were the same in patients with the Cuban disease as in patients with subacute myelo-optico-neuropathy (SMON), which had occurred in Japan nearly 30 years ago and from which Inoue-Melnick virus (IMV) type 1 was first isolated. We investigated the presence of IMV in cerebrospinal fluid samples of patients with this epidemic neuropathy in Cuba. METHODS: The established method for detecting IMV in MRC-5 fibroblasts was used throughout the study, which was conducted in two independent laboratories, one in Buffalo, NY, and the other in Kyoto, Japan. The cerebrospinal fluid samples of 20 patients and four controls were provided by Cuban researchers. RESULTS: All cerebrospinal fluid specimens from 20 Cuban patients tested were positive for IMV type 2 (100%), and four cerebrospinal fluid specimens from Cuban controls were negative for IMV (0%). These clear-cut results were identical in both laboratories. CONCLUSION: Our study indicates an important association of IMV type 2 with patients of this Cuban epidemic. The etiologic role of IMV type 2 in the epidemic neuropathy in Cuba, along with weak cytopathic effect viruses isolated by the Cuban group, remains to be elucidated.

Reappraisal of modified neutralization test for Inoue-Melnick virus.

Ito et al. (1992) reported an improved modification of the neutralization (NT) test for Inoue-Melnick Virus (IMV) by prolonged 90 min incubation of the mixture of the virus and serum. For confirmation, we compared the results of the NT test by 90 and 60 min incubation. Against human sera, the NT test by 90 min incubation was significantly more sensitive than the test by 60 min incubation. By 90 min incubation, the NT titers increased by 3 to 6 times and approximately 6% of antibody-negative adult sera previously determined in the tests by 60 min incubation turned to positive (more than 1:10). However, antibody-negative sera of Japanese children examined previously in the tests by 60 min incubation remained negative after the 90 min tests. On the other hand, against rabbit immune sera, no significant difference of NT titers was observed between the two NT tests for 90 and 60 min incubation. Also we reinvestigated Japanese adult sera for type 2 IMV infection by the NT test for 90 min incubation, but did not find any serum of type 2 or intermediate type infection. Therefore, it is not necessary to change the previous conclusion that in Japan IMV infections are mostly of type 1.

Direct isolation of Inoue-Melnick virus from human colorectal carcinoma tissues but not from stomach carcinoma tissues.

Inoue-Melnick virus (IMV) was isolated directly from all of the seven human colorectal adenocarcinoma tissues studied, whereas IMV was not isolated from five human stomach carcinoma tissues. Seven isolates from the Japanese colorectal carcinoma tissues were identified as type 1 IMV by the neutralization test and were sensitive to phosphonoacetic acid, a growth inhibitor of IMV. Furthermore, type 1 IMV-antibody was detected in six of seven sera of the patients whose colorectal carcinoma tissues were positive for virus isolation. In contrast, IMV-antibody was not detected in the five sera of the patients whose stomach carcinoma tissues were tested and found negative for IMV.

Detection of Inoue-Melnick virus (IMV)-antibody in Japanese patients with colorectal carcinoma and isolation of IMV from Japanese colorectal carcinoma cell lines.

The antibody prevalence rate of 3 types of IMV in 24 sera of Japanese patients with colorectal adenocarcinoma (mean age 60.8 years) was 83.3% (20/24). Of 20 positive sera, 19 contained type 1 antibody and one contained type 2. In contrast, the seropositivity of IMV in 21 sera of matched control patients (mean age 60.9 years) with non-colorectal carcinoma such as mammary and lung carcinoma was 23.8% (5/21). The 5 positive sera contained type 1 antibody. The seropositivity to IMV in 79 sera of healthy Japanese over 50 years of age (mean age 62.3 years) was 24.0% (19/79). Again all of the 19 positive-sera contained type 1 antibody. The seropositivity of IMV in the patients with colorectal adenocarcinoma was significantly higher than in the controls, non-colorectal carcinoma patients and healthy persons over 50 years of age (P less than 0.001). Type 1 IMV was isolated from each of 2 recently established Japanese colorectal adenocarcinoma cell lines.

5-Bromodeoxyuridine enhancement of Inoue-Melnick virus synthesis in human colon carcinoma cell lines.

Inoue-Melnick virus (IMV) synthesis in naturally infected human colon carcinoma cell lines can be enhanced by 5-bromodeoxyuridine (BUDR). The optimal conditions for BUDR enhancement differed between two cell lines, SW-480 and DLD-1. SW-480 cells produced 100 times more infectious IMV than DLD-1 cells by BUDR treatment. A clonal difference in IMV synthesis in SW-480 cells was also observed. The highest titer of virus was found in the culture fluid of clone H (SW-480/H) cells treated with BUDR, and a herpes-like virus was detected by negative-staining electron microscopy of concentrates of the culture fluid. One nonproducer clone (SW-480/B) was also activated to produce virus when treated with BUDR.

Isolation of Inoue-Melnick virus from human colorectal carcinoma cell lines.

Inoue-Melnick virus (IMV) was isolated from five of six human colorectal carcinoma cell lines, whereas the virus was not isolated from three normal colon-derived cell lines, three pancreas carcinoma cell lines, two bladder carcinoma cell lines, one kidney adenocarcinoma cell line, and one cervical carcinoma cell line. IMV synthesis was remarkably enhanced in the colorectal carcinoma cell lines by treatment with 5-bromodeoxyuridine (BUDR). However, virus was not detected in any of the other cell lines in spite of BUDR treatment. Five strains of virus isolated from colorectal carcinoma cell lines were subsequently identified by the neutralization test as intermediate-type IMV.

Enhancement of Inoue-Melnick virus synthesis by 5-bromodeoxyuridine in human meningioma (MG-1) cells.

The authors found remarkable enhancement of Inoue-Melnick virus (IMV) synthesis by 5-bromodeoxyuridine (BUDR) in human meningioma (MG-1) cells, a virus-producer line of type 1 IMV. Treatment with BUDR resulted in rapid and abundant synthesis of infectious IMV in MG-1 cells. The titer of the cell-associated virus in treated cells increased approximately 6.0 log 10 compared with that in untreated cells. Immunofluorescent antibody tests revealed that IMV-associated late antigen was induced by BUDR in the cytoplasm of approximately 50% of treated cells. A clonal difference was also found in the enhancement of BUDR on the IMV synthesis in MG-1 cells. The most remarkable enhancing effect of BUDR was observed in the clone C line, and a herpes-type virus was detected by negative-staining electron microscopy in the culture fluid of the clone C treated with BUDR.

Endogenous production of infectious Inoue-Melnick virus in a human meningioma cell line.

We investigated continuous production of Inoue-Melnick virus (IMV) in the MG-1 cell line, established from human meningioma. The infectious virus, identified as a type 1 virus, was mostly recovered extracellularly. Assay of MG-1 cells as infective centers indicated that most of the cells were capable of producing infectious virus. By immunofluorescence, more than 90% of the cells were found to have IMV-associated cytoplasmic antigen(s) (IMCA).

Factors influencing the assay of Inoue-Melnick virus.

We describe factors influencing the assay of Inoue-Melnick virus (IMV) and practical comments for its assay. MRC-5 cells at a population doubling level (PDL) of more than 32 were more sensitive than younger cells for demonstrating cytopathic effect (CPE) of the virus. The PDL of MRC-5 cells also influenced the virus yield, particularly when the amount of viral inoculum was small. Cells at younger PDL produced less virus. The appearance of CPE and the virus yield were inhibited by increasing the concentration of NaHCO3 to 0.14% in the maintenance medium. The release of the virus into the medium was also inhibited as NaHCO3 was increased.

Isolation of Inoue-Melnick virus from human meningioma-derived cell cultures and detection of antibody in patients with meningioma.

Inoue-Melnick virus (IMV) was isolated from six of seven human meningioma-derived cell cultures, while the virus was not isolated from six other brain tumor cell cultures. Sera of 145 consecutive neurosurgical inpatients were tested for IMV-neutralizing antibody. Of 26 patients with meningioma, 22 were positive for IMV antibody (84.6%). Of the remaining 119 patients, 16 were positive.

Antibody survey for Inoue-Melnick virus infections in Osaka and Houston.

Neutralizing antibodies to the Inoue-Melnick virus were detected in healthy adults in Osaka and in Houston. For adults, the prevalence rate for the cross-reacting type 3 virus was 13.8 per cent in Osaka and 29.1 per cent in Houston. Antibodies were also found in immune serum globulin prepared from American adults, but not in sera of Osaka children under 10 years of age. The Inoue-Melnick virus isolates so far obtained in Houston could be classified into three antigenic types, but in Japan all have been type 1. Type 3 antibody tests detect all homotypic infections plus about 82 per cent of type 1 infections. Thus, the actual prevalence rates for type 1 infections in Japan are somewhat higher than those found with the type 3 assay, namely, about 14 per cent for young adults and 24 per cent for those over 50 years of age. Further studies are needed to elucidate the natural history of this virus infection.

Thermostabilization of Inoue-Melnick virus by salt.

Inoue-Melnick virus (IMV) was stabilized to thermal inactivation at 50 degrees C by 1 M Na2SO4 but not by 1 M MgCl2, 1 M MgSO4, 2 M NaCl, and 2 M KCl. The latter salts enhanced the inactivation over that in distilled water. Na2SO4 stabilized IMV at 1 M concentration but enhanced inactivation at 0.1 M. Three types of IMV were stabilized by 1 M Na2SO4 at 50 degrees C to almost the same degree. The pattern of the thermostabilization of IMV by salts was similar to that of herpes simplex virus; however, the degree of its stabilization was lower with prolonged heating.

Inhibitory effect of acyclovir on the growth of Inoue-Melnick virus isolated from cerebrospinal fluid.

Inoue-Melnick virus (IMV) was isolated from the cerebrospinal fluid of patients with chronic neurologic disorders. The effect of acyclovir (ACV), 9-(2-hydroxyethoxymethyl)-guanine, on the growth of IMV was studied in human diploid cell cultures. ACV markedly inhibited the multiplication of all three IMV serotypes in vitro with the same inhibitory effect. By passage of IMV in the presence of ACV in the medium, ACV-resistant mutants of three IMV serotypes were easily isolated.

Inhibitory effect of glucocorticoid hormones on the growth of Inoue-Melnick virus.

Glucocorticoid hormones inhibited the early stage of propagation of Inoue-Melnick virus in a dose-dependent manner in human diploid cell cultures. Prednisolone and hydrocortisone achieved complete inhibition of replication of the three Inoue-Melnick virus serotypes at concentrations of 0.2 X 10(-4) and 10(-5) M, respectively; strong inhibition was found even at 10(-5) and 10(-6) M, respectively. The steroid hormones did not show a direct effect on the virus.

Classification of Inoue-Melnick virus into three antigenic types.

Antigenic analysis of Inoue-Melnick virus, formerly called SMON virus, showed that isolates from patients with subacute myelo-optico-neuropathy (SMON) in Japan, thus far tested, belonged to a single antigenic type (type 1). However, Inoue-Melnick virus isolates obtained so far in the United States chiefly from patients with multiple sclerosis could be classified into three types: type 1, type 2, and an intermediate type. At the present state of knowledge, the virus has not been proven to be the causative agent of disease, hence its provisional designation as IMV.

Isolation of virus from the spinal fluid of three patients with multiple sclerosis and one with amyotrophic lateral sclerosis.

A transmissible agent with the properties of a thermolabile filterable virus was isolated from the cerebrospinal fluid (CSF) of 4 Houston patients with chronic central nervous system (CNS) disease. 3 had multiple sclerosis and I had amyotrophic lateral sclerosis. No isolations were made from 27 patients whose CSF was taken for diagnosis of illness unrelated to degenerative CNS disease. Neutralising antibodies to the 4 isolates were present in the serum of each of the 4 patients with virus (14 positive results among 15 tests) but in hardly any of the sera from 8 patients without virus (3 antibody-positive results among 32 tests). The agent is antigenically related to the virus isolated in Japan from cases of subacute myelo-optico-neuropathy.