Takeo Matsumura

Cloning and structural analysis of the gene for cAMP-dependent protein kinase catalytic subunit from Plasmodium yoelii.

We isolated the gene encoding the cAMP-dependent protein kinase catalytic subunit (cAPK[C]) from Plasmodium yoelii by screening a genomic library for the DNA fragment as produced by the polymerase chain reaction. The deduced protein of 341 amino acids conserves residues that are important for the function of serine/threonine protein kinases and shows the highest homology to cAPK[C]s of other organisms. However, P. yoelii cAPK[C] has 8 residues, which are perfectly conserved in cAPK[C]s of other organisms, radically replaced with residues having different side-chain properties. It is stressed that two radical replacements occur in regions for the binding with a regulatory subunit and/or a heat-stable inhibitor protein.

Effect of intestinal helminth infection on retinol and β-carotene status among rural Nepalese.

Intestinal helminth infection and its effect on vitamin A, retinol and β-carotene, was studied in 224 [145 children (aged less than 15 years) of Okharpauwa Village Development Committee (VDC) (Nuwakot district) and 79 inhabitants (mainly adults) of Boya VDC (Bhojpur district)] subjects living at an altitude of 2000 m. Direct smear technique in duplicate was applied to detect helminth eggs. Most common helminth detected was Ascaris lumbricoides followed by Trichuris trichiura in Okharpauwa VDC and by hookworm in Boya VDC, respectively. Mixed helminth infections were relatively low (7.3% in Okharpauwa VDC and 11.1% in Boya VDC). The retinol and β-carotene were estimated by high-performance liquid chromatography (HPLC). The helminth eggs positive children of both sexes in Okharpauwa VDC had significantly lower serum retinol concentration compared with their helminth eggs negative counterparts (P 0.05) but in β-carotene level (P <0.05). Results suggest that deworming contribute significantly in the prevention of vitamin A deficiency associated morbidity and mortality among children in these intestinal helminth prevalent rural communities in Nepal.

Transovarial Transmission of Chikungunya Virus by Aedes albopictus Mosquitoes Ingesting Microfilariae of Dirofilaria immitis under Laboratory Conditions

Female Aedes albopictus mosquitoes of the Miki strain were experimentally fed on defibrinated sheep blood containing 5× 107 PFU of chikungunya virus and 20,000 microfilariae of Dirofilaria immitis per milliliter. Fully engorged mosquitoes transmitted the virus to a small percentage of the F1 progeny, but females of the F1 generation did not transmit the virus to the F2 progeny. The control mosquitoes that ingested the virus without microfilariae did not transmit the virus to their eggs, larvae, or pupae in the F1 or F2 generations. These results showed that A. albopictus of this strain that concurrently ingested the virus and microfilariae transmitted the virus by the transovarial route under experimental conditions.

Morphogenesis of Dengue-1 Virus in Cultures of a Human Leukemic Leukocyte Line (J-111)

In previous experiments we found that cultures of a human leukemic leukocyte line ( J-111) supported good growth of dengue-1 virus (K. Shiraki and S. Hotta, unpublished data). In this communication we describe results of electron microscopic observations on the infected J-111 cells. Two strains of dengue-1 virus were used: Mouse-passaged Mochizuki strain, and strain 32748 propagated in Aedes albopictus mosquitoes (kindly supplied by Dr. L. Rosen, Pacific Research Section, NIH, Hawaii) (11). The former was in the form of infected mouse brain homogenate, and the latter was of mosquito emulsions

Studies on the cryopreservation of eggs of Angiostrongylus cantonensis

The possibility of cryopreserving the eggs of Angiostrongylus cantonensis collected from the uterus of female worms was investigated. Eggs were cultured in NCTC 109 medium containing 50% rat serum, and various growth stages, from one-cell eggs to embryonated eggs, were used in this study. As a cryoprotective agent, dimethylsulphoxide (Me2SO) was added to the medium at a final concentration of 1 M. Eggs suspended in 0.2 ml of the medium at 37 degrees C were cooled to 0 degrees C at a rate of 1 degree C min-1, then an equal volume of 2M-Me2SO solution was added. After equilibration for 15 min, the freezing procedures were started. In the freezing procedures, the effectiveness of (i) a seeding process, (ii) different cooling and warming rates and (iii) the relationship between the growth stages of the eggs and their tolerance to freezing at -20 degrees C were investigated. It was found the highest level of survival could be obtained with 32-cell eggs cooled at a rate of 0.3 degrees C min-1 or more slowly with seeding at -4 degrees C and warming at a rate of 5 degrees C min-1. Survival was influenced more by cooling rate than by warming rate. Using these optimum conditions, the survival of eggs was then investigated following cooling to various temperatures. While more than 50% of eggs were found to survive cooling to -30 degrees C, extremely low survival was noted from lower temperatures.

Release of Arboviruses from Cells Cultivated with Low Ionic Strength Media

Discussion and Summary The present study indicates that production of CHIK and DEN-1 viruses in BHK-21 cells were inhibited by lowering NaCl concentrations of culture media. The inhibitory effect was reversed rapidly (within 30 sec) by placing the cells in normal medium. By changing the media from normal to low NaCl concentrations, the inhibition of virus release again took place within short periods of time. In this manner, the inhibition of virus release and its recovery could be repeated practically indefinitely in a particular culture. The effect was due to the ionic strength rather than osmolarity of media. No substances other than the salt such as amino acids or culture medium ingredients were regarded to be involved. While the present data are compatible with those previously reported with viruses of Sindbis (3) and polio (2), some unique findings were obtained in our electron microscopic studies. The cells cultured in low ionic strength media did not reveal characteristic pictures of viral budding from the cell surface membrane as commonly seen in cells cultured in normal media. In spite of it, the “precursor particles” (8, 9) were observed in the cells cultured in low ionic strength media as in normally cultured cells. As generally accepted (7-12), the group A arboviruses acquire their outer coat from the cell surface membrane of host cells during the budding stage. The ionic strength seems to influence this process, altering certain biochemical and/or biophysical conditions of the cell membrane structures. This may have a significant relation with the mechanism of virus release from host cells such as observed with Western equine encephalitis virus in chick embryo cells (13) or Venezuelan equine encephalitis virus in KB cells (14).

Increased Multiplication of Dengue Virus in Mouse Peritoneal Macrophage Cultures by Treatment with Extracts of Ascaris-Parascaris Parasites

Methylcellulose‐elicited peritoneal macrophages from BALB/c mice were cultivated in vitro and inoculated with dengue virus (DV). At intervals thereafter portions of the culture fluids were taken and titrated for viral infectivity. Extracts from Ascaris suum and Parascaris equorum, either crude or Sephadex G‐100 fractionated, were examined for effects on the multiplication of DV. The macrophage cultures treated with the above substances produced larger amounts of DV compared with untreated control cultures. The enhancing effect of the substances depended on doses added and duration of treatment and was suppressed by co‐treatment with carrageenan, a specific macrophage‐inhibiting agent, but was not related to the viability of cultured cells. In fluorescent antibody (FA) as well as infectious center assay experiments, it was shown that the DV‐infected cells were found more frequently in treated cultures than in untreated control cultures. In the treated cultures phagocytosis by cultured cells was also of a higher magnitude than that in untreated cultures. In cocultures of macrophages and splenocytes from the same line of mice, no additive effect of splenocytes was noted. The limulus amebocyte lysate clotting enzyme reaction (Limulus test) indicated that involvement of bacterial lipo‐polysaccharides in the enhancement phenomena was negligible. The data so far obtained suggest that the enhancing effect was due to direct action of the parasitic extracts on macrophages. Four Sephadex G‐100 fractions from the crude extracts showed similar activities; however, the effects of fractions I and III appeared to be comparatively strong. Significance of the findings in relation to the pathogenesis of DV infection was discussed.

Effects of Ca++ Ion on the Liberation of Dengue Virus from BHK‐21 Cells in Culture*

The mechanism by which virus is liberated from infected cells is important for studying the process of virus infection. At least three possible ways of virus liberation are (i) reverse phagocytosis (exocytosis), (ii) disruption of cells (bursting) and (iii) budding from the cellular membrane. As for dengue viruses, there have been a few reports suggesting the exocytotic discharge of virus based on electron microscopic observations (1, 2, 5). It has already been reported that a high Mg++ concentration in a media of dengue-infected Vero cells enhances virus liberation from the cells (7). The mechanism underlying this phenomenon may probably be stimulation of the liberation of mature virus from the intracytoplasm to the extracytoplasmic space under the influence of the high Mg++ concentration in media (8). These facts prompted us to study the Ca++ dependency of dengue virus liberation from infected cells, which suggested that this phenomenon was due to an exocytotic process. A concept has been presented that exocytosis is the mechanism for the release of secretory substances from neuron, neurosecretory, endocrine, and mast cells and that Ca++ ion is essential for the release of secretions by this exocytotic process (10). If the virus liberation from cells is due to exocytosis, it might be possible that Ca++ ion dependency exists. We examined this possibility in the present work. Type 1 dengue virus (Mochizuki strain) (DEN) and, as a control, chikungunya virus (African strain) (CHIK) were used. It has been known that chikungunya virus is liberated from the cell surface membrane by the process of budding instead of exocytosis (4,6). We studied whether or not maintenance of cells in a Ca-free culture medium containing EDTA, a chelating agent, affected DEN liberation.

Amino Acids Requirements for Reproduction of Arboviruses

Discussion and Summary In the present experiments, it was shown that the maturation of CHIK and DEN-1 viruses was markedly suppressed by the deprivation of glutamine and cystine, respectively. The difference of amino acid requirements may reflect a difference in the maturation process of the two kinds of viruses. Evidence have been presented, indicating that the maturation sites of group A and B arboviruses were characteristically different from each other (11, 14). Some details of the mechanism(s) of the phenomena were investigated, especially those dealing with CHIK virus and glutamine. The inhibitory effect of deprivation was reversible; the virus yields were restored by putting the cells from deprived medium into normal medium and vice versa. Such restoration and suppression were particularly clear during the earlier stages of the virus infection. In the radioisotope incorporation experiments, it was shown that the deprivation was not directly related to the viral RNA or protein synthesis in the infected cells. In the electron microscopic examinations, the cells infected with virus and cultured with deprived medium did not reveal the “core particles” inevitably seen in the same cultures fed with normal medium. This finding suggests that the deficiency influences the step(s) prior to the appearance of the core particles. This may be compatible with the above-mentioned data indicating that the reversibility of the deficiency effect was more clearly demonstrated during the earlier stages of the virus infection than in the later stages. It can be postulated therefore that the amino acid deprivation (L-glutamine in the case of CHIK virus) is possibly involved at certain stages for the assembly of viral structures to produce complete virions. Since, however, it was clearly shown that the glutamine did not compensate the low ionic strength effect already described, the viral maturations stages affected by amino acid deficiency and low ionic strength are probably different from each other.