Kil-Won Kwon

Photoinactivation of Virus Infectivity by Hypocrellin A

Abstract— We investigated the photoinactivation of virus infectivity by hypocrellin A and its mechanism. The titers of vesicular stomatitis virus (VSV) and human immunodeficiency virus type 1 (HIV‐1), both of which are enveloped viruses, were reduced upon illumination with hypocrellin A in a concentration‐dependent manner, whereas canine parvovirus, a nonenveloped virus, was not killed. The removal of oxygen or addition of sodium azide or bT‐carotene both inhibited VSV inactivation. Mannitol and superoxide dismutase had no effect on VSV inactivation. These results indicate that singlet oxygen was involved in the process of VSV inactivation. Of the three major VSV membrane proteins, peripheral membrane protein M was most damaged by the hypocrellin A phototreatment.

Growth-blocking peptide titer during larval development of parasitized and cold-stressed armyworm

Growth-blocking peptide (GBP) has been isolated for the first time from the haemolymph of the host armyworm Pseudaletia separata whose development was halted in the last larval instar stage by parasitization with the parasitoid wasp Cotesia kariyai. Recent studies demonstrated that GBP not only exists in the plasma (haemolymph without cells) of parasitized last instar larvae, but also in the plasma of nonparasitized penultimate (5th) instar larvae. Monoclonal antibodies were prepared to measure the titers of GBP in nonparasitized and parasitized larval plasma. One of three monoclonal antibodies raised against GBP, which is the most specific for GBP, was used to quantify the concentration of plasma GBP. As this antibody recognized two plasma peptides other than GBP in crude plasma fractions, each plasma peptide fraction was separated by a reversed phase HPLC, and then plasma GBP level was measured by ELISA. The highest level of plasma GBP detected on Day 0 of the penultimate instar larvae was gradually decreased throughout the larval growth except for the temporary increase on Day 0 of last larval instar. After parasitization on Day 0 of last larval instar, two peaks of plasma GBP titer were detected during the last larval instar, one day and six days after parasitization. This characteristic increase and decrease in plasma GBP level was also observed by transferring last instar larvae of the armyworm from 25 to 10 degrees C, as a result of which larvae delayed pupation by more than 15 days. From these results, it is reasonable to propose that plasma GBP in lepidopteran larvae might control certain upstream steps in a cascade of events leading to pupation; thus, an elevated level of plasma GBP interferes with normal metamorphosis from larvae to pupae.

Human Cytomegalovirus DNA Is Not Detectable with Nested Double Polymerase Chain Reaction in Healthy Blood Donors

The PCR method was introduced to detect cytomegalovirus (CMV) DNA from 189 peripheral blood samples of volunteer donors. We adopted the nested double PCR method with primers specific for immediate early gene 1 followed by electrophoresis and ethidium bromide staining. This nested double PCR method was sensitive enough to detect approximately a single copy of CMV DNA. However, we failed to obtain positive amplification of CMV DNA from any of these donor samples. In contrast, CMV DNA could be detected in all 3 tested immunocompromised patients who had undergone bone marrow transplantation. These results support our previous report that the frequency of CMV DNA is of an order lower than 1 copy/105 leucocytes in the peripheral blood of healthy seropositive individuals.

A Human Monoclonal Antibody to High‐Frequency Red Cell Antigen Jra

A human‐mouse heterohybridoma (HMR0921) secreting human monoclonal IgG3, Λ antibody was produced from peripheral blood lymphocytes of a healthy blood donor with serum antibody to Jra, by EBV transformation and hybridization with mouse myeloma cell line P3X63Ag8.653. The reactivity of HMR0921 antibody was assessed by antiglobulin test with a panel of red cells including 14 different rare blood types. Only Jr(a‐) red cells were negative. The strict specificity of this antibody to Jra antigen was further confirmed by absorption test with fluorescence flow cytometry. On screening of 28,744 blood donor samples by HMR0921 antibody, we detected 19 agglutination‐negative samples, which were confirmed as Jr(a‐) by conventional anti‐Jra antisera. Therefore, our HMR0921 antibody is extremely useful for detecting rare Jr(a‐) blood.

Oligo-2',5'-adenylate synthetase activity in cells persistently infected with human T-lymphotropic virus type I (HTLV-I)

Spontaneous production of interferon‐gamma (IFN‐γ) was shown in several T‐lymphoblastoid cell lines persistently infected with human T‐lymphotropic virus (HTLV‐1). However, the produced IFN‐γ was not always associated with the induction of the antivirus state. The induction of oligo‐2′,5′‐adenylate synthetase (2‐5AS) by IFN was studied in five human T‐cell lines persistently infected with HTLV‐I (MT‐1, MT‐2, SMT‐1, HUT 102 and OKM‐2). Four cell lines are able to produce IFN‐γ spontaneously, while the OKM‐2 cell line is not. Poor induction of 2‐5AS was recognized in three (MT‐1, MT‐2 and SMT‐1) of the four cell lines producing IFN‐γ, though the poor induction was improved after long‐term cultivation of cells with IFN‐α. On the contrary, in the OKM‐2 cell line. significant activity of the enzyme was induced by IFN‐α. Induction of 2‐5AS was not correlated with cell growth inhibition, but with the antivirus state. Furthermore, an inverse relationship between IFN‐γ production and 2‐5AS induction was demonstrated in these cell lines with the exception of HUT 102 cells.

Preparation of Virus-Free Pyridoxylated Hemoglobin from the Blood of HBV or HTLVI Healthy Carriers

One of our major aims was to find ways to utilize outdated or virus-contaminated blood. Pyridoxylated hemoglobin (PLP-Hb), a possible substitute for red cells as an artificial oxygen carrier was prepared from outdated human blood. By conjugation with polyethylene glycol (PEG), the biological half life was increased about 3 folds at 82% blood replacement in rats without significant side effects in vivo or in vitro. We next tried to prepare virus-free PEG-PLP-Hb from HBV or HTLV-I positive blood. A considerable amount of HBV (Dane particles) could be removed from HBV-positive red cells by washing and during the preparation of PEG-PLP-Hb. When the hemoglobin preparations containing Dane particles were filtered through a porous cellulose filter, BMM-30 (30 nm pore size, Asahi Chemical Industry Co., Ltd., Osaka, Japan), HBV-DNA in the filtered fractions became less than 0.33% of the initial amount. More than 96% of blood leukocytes could be removed with a leukocyte removal filter, Sepacell R-500 (Asahi Medical Co., Ltd., Tokyo, Japan). The leukocytes collected from filtrated fractions of HTLV-I positive blood did not survive beyond 3 days. Since transmission of HTLV-I occurs by cell to cell contact and is rare in cell-Free condition, it is unlikely that the PLP-Hb prepared from HTLV-I positive blood which is deprived of leukocytes transmits HTLV-I infection.

Evaluation of the Human T‐Cell Leukemia Virus Type I Seropositivity of Blood Donors by the Particle Agglutination Inhibition Test

In the HTLV‐I seroscreening of blood donor sera by gelatin particle agglutination (PA), more than 50% (55.6%) of the PA‐positive sera were negative by immunofluorescence assay (IF). However, when donors were divided into age groups, there were increasing numbers of IF‐positive/PA‐positive donors with age. Among the PA‐positive donors in the 50–64 age group, 65.9% were IF‐positive compared to 16.0% in the 16–19 age group. The serological specificities of the IF‐negative/ PA‐positive specimens were tested by using a newly developed PA inhibition (PAD test. The HTLV‐I specificity of the PAI test was confirmed by the observation that agglutinations with anti‐HTLV‐I p19 and gp21 monoclonal antibodies as well as IF‐positive sera were specifically inhibited with HTLV‐I preparations or HTLV‐I‐positive cell extracts and not with HTLV‐I‐negative cell extracts. Sixty of the 104 specimens collected randomly from the IF‐negative/PA‐positive donors were PAI‐positive. The majority (80%) of such PAI‐positive sera showed more than two bands of HTLV‐I gag‐encoded polypeptide, p19, p24, p2S and p53 on Western blotting. Some of the PAI‐positive sera were also positive by enzyme immunoassay. These results indicate that at least some of the IF‐negative/ PA‐positive donors possess HTLV‐I‐specific antibody and may be potential HTLV‐I carriers who will become IF‐positive at a later age