Survey of Japanese encephalitis virus in pigs and wild boars on Ishigaki and Iriomote Islands in Okinawa, Japan.

SUMMARY We previously revealed that Japanese encephalitis virus (JEV) seroprevalence was 4.5% in pigs on Ishigaki Island from 2005 to 2007. However, a partial E gene sequence (151 bp) of the JEV genome (JEV/sw/Ishigaki/1/2005) was detected in one pig. Phylogenetic analysis showed that JEV/sw/Ishigaki/1/2005 belonged to genotype III and to the same lineages isolated in Taiwan from 2006 to 2008. Serum samples were collected from 128 pigs on Ishigaki from 2009 to 2010, 24 wild boars on Ishigaki from 2008 to 2010, and 117 wild boars on Iriomote Island from 2008 to 2010. Four (3.1%) pigs on Ishigaki were positive for JEV antibody, but all wild boars on the island were negative. Fifty-two (44.4%) wild boars on Iriomote were positive for JEV antibody, in contrast to a seroprevalence of 3.7% in 2000 and 2004. JEV on Iriomote and/or in Taiwan might be related to transmission on Ishigaki.

Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.

AIMS: To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. METHODS: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. CONCLUSIONS: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE.

Confirmation of dengue virus infection by detection of dengue virus type 1 genome in urine and saliva but not in plasma.

We successfully detected dengue virus (DENV) genome in urine and saliva but not in plasma samples from a Japanese dengue fever patient. The results of the present study suggest that detection of DENV genome in urine and saliva can be an effective diagnostic method, particularly for children with viral hemorrhage.

Molecular characterization of S locus genes, SLG and SRK, in a pollen-recessive self-incompatibility haplotype of Brassica rapa L.

In Brassica species that exhibit self-incompatibility, two genes, SLG and SRK, at the S locus are involved in the recognition reaction with self and non-self pollen. From a pollen-recessive S29 haplotype of Brassica rapa, both cDNA and genomic DNA clones for these two genes were isolated and characterized. The nucleotide sequence for the S domain of SRK29 showed a high degree of similarity with that of SLG29, and they belong to Class II type. RNA gel blot analysis showed that the transcript of SLG29 consisted of the first and second exons, and no other transcript containing any part of the intron sequence was detected. Because no transmembrane domain was encoded by the second exon of SLG29, SLG29 was designated a secreted type glycoprotein. SLGs of two other pollen-recessive haplotypes, S40 and S44, of B. rapa also had a similar structure to that of SLG29. Previously, SLG2 from a pollen-recessive haplotype, S2, of Brassica oleracea was found to produce two different transcripts, one for the secreted type glycoprotein and the other for a putative membrane-anchored form of SLG. Therefore, the nature of these SLGs from pollen-recessive haplotypes of B. rapa is different from that of SLG2 of B. oleracea.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

Human antibodies responsible for binding inhibition and polymerization inhibition of human immunodeficiency virus type 1 reverse transcriptase.

Using a solid-phase non-radioisotopic (non-RI) reverse transcriptase (RT) assay, antibodies inhibiting human immunodeficiency virus type 1 (HIV-1) RT activity (RTI antibody) were investigated for their ability to inhibit binding of RT to a template-primer and DNA polymerization. The RTI antibody inhibited the binding of RT to the template-primer (BI antibody), and directly reacted with the RT-template-primer complex and inhibited enzymatic activity (PI antibody). The RTI antibody interfered with formation of the RT-template-primer complex suggesting that it recognized the antigenic site involved in template-primer binding of RT molecules. Since deoxynucleotide triphosphates (dNTPs) blocked inhibition of the RT activity by the PI antibody, the antigenic site recognized by the PI antibody may be closely related to the dNTP binding site. The seropositivities of the BI and PI antibodies were 84.6% and 91.2%, respectively, in HIV-1-infected individuals; healthy individuals, HTLV-I-positive individuals, autoimmune disease patients and leukemia patients were all seronegative. No significant correlation of residual RT activities was observed when BI and PI antibodies were compared (r = 0.688). It is possible that the epitopes recognized by the BI antibody differs from those recognized by the PI antibody. The assays described are able to detect BI and PI antibodies in the sera of HIV-1-infected individuals.

T-cell activation and induction of antibodies and memory T cells by immunization with inactivated Japanese encephalitis vaccine.

Mouse brain-derived inactivated Japanese encephalitis (JE) vaccine is the only currently internationally accepted vaccine against JE virus. We analyzed cellular and humoral immune responses to the JE vaccine in healthy adults in order to understand the protective immunity induced by this vaccine. Immunization with the JE vaccine induced T-cell activation in vivo, demonstrated by increase in the plasma levels of interleukin (IL)-2 and soluble CD8. JE virus-specific antibodies determined in radioimmunoprecipitation (RIP), hemagglutination inhibition (HI), and neutralization assays were also induced by immunization with the JE vaccine. JE virus-specific memory T cells were detected 60 days after immunization. These results suggest that protective immunity induced by the inactivated JE vaccine includes JE virus-specific T cells as well as antibodies with multiple biological activities.

Expression of p53 protein in benign epithelial hyperplasia, atypical ductal hyperplasia, non-invasive and invasive mammary carcinoma: an immunohistochemical study.

To clarify whether p53 protein expression is involved in multistep carcinogenesis or the progression of mammary ductal carcinoma, we investigated p53 protein expression in 83 invasive ductal carcinomas (IDC), 10 IDC with a predominant intraductal component, 13 non-invasive ductal carcinoma (NIDC), 16 atypical ductal hyperplasia (ADH) and 39 benign epithelial hyperplasia (EH), using immunohistochemistry. Expression of p53 protein was detected in 24 (28.9%) cases of IDC, 5 (50%) cases of IDC with a predominant intraductal component and 1 (7.6%) case of NIDC. No expression was observed in either ADH or EH. In IDC, including cases with a predominant intraductal component, p53 protein expression was associated with a higher histological grade (P < 0.0001) or mitotic index (P < 0.0005). Although overexpression of c-erbB-2 protein has also shown a similar association with these prognostic indicators, expression of p53 protein correlated regardless of the status of c-erbB-2 overexpression. Completely coordinated expression of p53 protein was seen in both intraductal and invasive components. The intraductal component in IDC including cases with a predominant intraductal component which expresses p53 protein had significantly higher histological grade (P < 0.0005) or more comedo-subtypes (P < 0.0001). These results suggested that p53 protein expression occurs at a stage of NIDC with high histological grade or in comedo-subtypes. Its expression is maintained throughout invasion.

Development of dengue IgM-capture enzyme-linked immunosorbent assay with higher sensitivity using monoclonal detection antibody.

An IgM-capture enzyme-linked immunosorbent assay (IgM-ELISA) is used widely for serodiagnosis of dengue. A dengue IgM-ELISA with higher sensitivity has been developed. In the new ELISA, anti-dengue IgM antibody, which had been captured on the solid phase, was reacted with tetravalent dengue viral antigens, and detected by a flavivirus group specific monoclonal antibody, D1-4G2-4-15 (4G2). Reaction of 4G2 to viral antigens was similar to that of dengue patients' IgG. Non-specific reaction of 4G2 to the control antigen, which was prepared from uninfected cell culture fluid of mosquito C6/36 cells, was much lower than that of patients' IgG. Thus, specificity of the ELISA with 4G2 was much higher than that with patients' IgG, and lower levels of specific IgM was detected in the serum samples. These results suggest that the modified dengue IgM-ELISA with monoclonal antibody 4G2 has many advantages over the original "in-house" ELISA.

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.

A case of spindle cell sarcomatous change of hepatic ducts manifesting as obstructive jaundice.

Spindle cell carcinoma is a rare tumor commonly occurring in the upper aerodigestive tract. We report a 62-year-old male with spindle cell sarcomatous change located at the hepatic hilum, resulting in obstructive jaundice. The patient died after an extended resective operation. The rare disease and its histogenesis is discussed.

Immunohistochemical studies of S-100 protein expression in myoepithelial cells of benign breast diseases and normal breast tissues.

We studied benign breast diseases using polyclonal anti S-100 protein and monoclonal anti a-subunit or beta-subunit of S-100 protein antibodies. The antibody gave positive staining in most of the cytoplasms and nuclei of the myoepithelial cells of normal breast tissues, gynecomastia, fibroadenoma, intraductal papilloma and mastopathy. However, immunohistochemical methods using the monoclonal antibodies for them did not reveal positive staining in the myoepithelial cells.

Chronic toxicity and tumorigenicity study of aluminum potassium sulfate in B6C3F1 mice.

The tumorigenic potential of aluminum potassium sulfate [A1K (SO4)2 12H2O, APS], a compound which exists widely in the environment, was investigated in B6C3F1 mice. APS was administered in the diet for 20 months at dose levels of 1.0, 2.5, 5.0 and 10.0% (w/w). One group receiving the basal diet served as the control. Body weight gain in both sexes was decreased in the 10.0% APS treated group, and increased in the 1.0 and 2.5% APS treated groups. The survival rates at the end of the dosing period were 73.3% (male) and 78.3% (female) in the control group, and 86.7-95.0% (male) and 86.7-91.7% (female) in the APS treated groups. The survival rate showed a tendency to increase in both sexes in all the APS treated groups. In the tumor pathology, the incidence of hepatocellular carcinoma was significantly decreased in the males in the 10% APS treated group. The incidence of hepatocellular carcinoma was significantly decreased in females in all groups including the control group. As regards the nontumorous pathology, the incidence of myocardial eosinophilic cytoplasm showed a significant dose-dependent decrease in males in the APS treated groups. A comparison between the sexes revealed a significant decrease in the incidence of hepatocytic anisonucleosis, myocardial eosinophilic cytoplasm and acinar cell vacuolation of the submandibular gland in the females; and lymphocyte infiltration in renal cortex and pelvis, and vacuolation of cerebellar white matter were noted in the males. The results of the present study indicate that long-term administration of APS does not exert tumorigenic or any other toxic actions in B6C3F1 mice.

Laboratory diagnosis of dengue virus infection by reverse transcriptase polymerase chain reaction (RT-PCR) and IgM-capture enzyme-linked immunosorbent assay (ELISA).

Dengue virus infections are a major public health problem in most tropical and sub-tropical countries of the world. Dengue is occasionally imported by travelers who visit tropical areas and become infected with dengue virus. Laboratory diagnosis is essential for confirming the diagnosis of this virus. For purposes of confirmation, detection of specific IgM by IgM-capture enzyme-linked immunosorbent assay (ELISA) and of dengue virus genome by reverse transcriptase polymerase chain reaction (RT-PCR) have recently been used. In the present study, we tested serum specimens from dengue-suspected Japanese cases, by IgM-capture ELISA, RT-PCR, HI, and virus isolation. Serum samples collected before or on the day of defervescence were positive by RT-PCR, though no PCR-positive samples were obtained after fever day 1. IgM-capture ELISA was positive as early as disease day 4, and all samples but one were IgM-positive when collected on disease day 5 or later. In light of these findings, we recommend that both RT-PCR and IgM-capture ELISA be performed, irrespective of the stage of dengue illness. Combination of RT-PCR and IgM-capture ELISA increases the ability to diagnose dengue virus infection, even in the only that a single serum specimen from the patient is available.

Mitral valve replacement in ischemic mitral regurgitation. Preservation of both anterior and posterior mitral leaflets.

BACKGROUND: The surgical risks associated with ischemic mitral regurgitation are thought to be greater than those for other forms of mitral regurgitation. We have performed mitral valve replacement using the St. Jude Medical bileaflet prostheses with preservation of both leaflets, along with all of the chordae tendineae and papillary muscles. The aim of this study was to retrospectively evaluate mitral valve replacement with preservation of both mitral valves with respect to long-term clinical results and left ventricular performance. METHODS: Between January 1, 1988 and February 29, 2000, 15 patients were operated on for ischemic mitral regurgitation. There were 7 males and 8 females, and the mean age was 69.7+/-8.1 years. The preoperative variables showed clinical deterioration of the state, such as emergency operation in 40% of the patients, more than NYHA functional III class in 93% of patients, cardiogenic shock in 47% of the patients, a mean left ventricular ejection fraction of 36.8%, and a mean left ventricular end-systolic volume index of 116.7 ml/m2. RESULTS: There were 5 (33.3%) hospital deaths during the follow-up period including 1 early death and 1 (10%) late death during the follow-up period. Thus, the actuarial survival rate after 5 years for the whole was 60%. However, the left ventricular dimensions and left ventricular fractional shortening, even if in patients with profound depressed left ventricular function preoperatively, showed maintenance of the cardiac function. CONCLUSIONS: These results suggested that mitral valve replacement using the St. Jude Medical prostheses with preservation of both leaflets and all chordae tendineae and papillary muscles might be a procedure of choice for ischemic mitral regurgitation.

Detection of rabies virus RNA isolated from several species of animals in Brazil by RT-PCR.

Brain samples from different animal species including humans: five vampire bats, 14 cattle, 12 dogs, 11 cats, two horses, one pig, one sheep and three humans collected from various geographical regions of Brazil were found to be positive for rabies by means of the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). The brain samples were retested for rabies by means of the reverse transcription and polymerase chain reaction (RT-PCR) with 2 primer sets (P1/P2 and RHNI/RHNS3), which amplified full or partial regions on the nucleoprotein (N) gene of the rabies virus, respectively. Brain samples from five vampire bats, 13 cattle, one horse and one sheep failed to yield PCR products when the RHN1/RHNS3 primer pair was used, but all brain samples successfully yielded the products when the P1/P2 primer pair was used. These results suggest that Brazilian rabies virus isolates could be principally divided into two populations according to genetic difference.

Inhibitory effects of selenium, vitamin A and butylated hydroxytoluene on growth of human maxillary cancer cells in vitro.

The effects of vitamin A, selenium, and butylated hydroxytoluene (BHT) on the growth of a human maxillary cancer cell line were examined in monolayer cell cultures. The colony-forming assay showed a 50% reduction in the survival rate of the cell line at a concentration of 3.6 micrograms/ml of selenium, 28 micrograms/ml of vitamin A, and 74 micrograms/ml of BHT. Flow cytometric analysis with both FITC-labeled bromodeoxyuridine monoclonal antibody and propidium iodide demonstrated an increase of the S-phase fraction in the presence of selenium, an increase of the G0/G1-phase fraction in the presence of vitamin A, and an increase of the G2-M-phase fraction 1 day followed by an increase of G0/G1-phase fraction from the 3rd to 7th day when BHT was added. These results suggest that the mechanisms of inhibition of DNA synthesis by these compounds are different.

Predetermining postoperative hepatic function for hepatectomies.

To perform major resections safely in liver cancer operations, we have developed a reliable preoperative method to estimate postoperative liver function. We first conducted a series of studies using dogs to determine the various correlations between the volume of the remaining liver and postresectional hepatic function. We calculated Indocyaningreen (ICG) plasma disappearance and retention rates 15 minutes after intravenous injection of ICG at 0.5 mg/kg and found a 1:1 ratio, thus: ICG(K) postop. = ICG(K) preop. x functional remaining rate. This was applicable in our study of dogs with normal livers; in clinical cases complicated by liver cirrhosis and cancer, however, the functional rate of the remaining liver cannot be calculated only by the volumetric ratio of the whole liver and the remaining part. In clinical cases it must also be calculated by using hepatic vein catheterization and by measuring the ICG extraction rate of each segment of the part to be resected and the part to remain. By using this segmental liver function test, the postoperative hepatic function can be estimated accurately before the operation, thus enabling the surgeon to perform major hepatectomies safely and efficiently.