Susumu Hotta

Effects of Tannic Acid and Its Related Compounds upon Chikungunya Virus

The present report describes not only the effects of tannic acid (TA; belonging to hydrolyzable tannins) and its related compounds upon the infectivity of Chikungunya virus (CHIKV) but also the mechanism involved in this phenomenon. Our data show that TA inactivates CHIKV in vitro. Since the inactivating effect turned out to be pH‐dependent and was suppressed by bovine serum albumin, it is most probable that the virus‐inactivating capacity of TA is attributable to its preferential binding to proteins of virus particles. Examination on the virus‐inactivating capacities of some TA‐related compounds and comparison of their structures indicated that the active site of TA and its analogues might be the phenolic hydroxyl groups in their molecules. It seems that the active groups interact with the proteins of virus particles, resulting in a reduction or loss of viral infectivity. Discussion is made on the specificity of the actions of tannins and the possibility of application thereof to chemicals which are useful to investigate the nature and properties of viral proteins.

Dengue virus multiplication in cultures of mouse peritoneal macrophages: effects of macrophage activators.

Dengue‐2 virus multiplied in cultures of methylcellulose‐induced peritoneal macrophages of BALB/c mice. The in vitro‐cultivated macrophages from dengue‐1 virus‐immune mice produced larger amounts of dengue‐2 virus than did those from nonimmune controls. The effect of macrophage activators was examined by using nonimmune macrophages. Enhanced virus production was demonstrated in cultures of macrophages pretreated with phytohemagglutinin (PHA) or bacterial lipopolysaccharide (LPS). The number of virus‐infected cells in the pretreated cultures was estimated to be about 0.01% or less of the total macrophages. Continuous treatment of macrophages with PHA before and after virus inoculation brought about the most marked enhancement of dengue‐2 virus multiplication. On the other hand, treatment with concanavalin A or pokeweed mitogen showed little effect on the multiplication of the same virus. Treatment with carrageenan, a specific macrophage blocking agent, markedly suppressed dengue‐2 virus production in both dengue‐1 virus‐immune macrophages and LPS‐treated macrophages. The indirect fluorescent‐antibody (FA) technique revealed dengue‐2 viral antigen in the cytoplasm of infected macrophages, and the FA‐positive macrophages were more numerous in PHA‐treated cultures than in untreated controls. The results obtained are discussed in relation to a possible role of activated monocytes/macrophages in the pathogenesis of dengue hemorrhagic fever.

Dengue Type 2 Virus Infection in Human Peripheral Blood Monocyte Cultures

Dengue type 2 virus (D2V) infection in cultured human monocytes was studied. D2V permissiveness of the monocytes was enhanced when the cells were inoculated with D2V in the presence of either polyclonal or type‐specific monoclonal anti‐dengue antibody. The enhancement of D2V permissiveness mediated by the antibodies was more clearly demonstrated when the monocytes had been treated with trypsin before virus inoculation, though treatment of the cells with trypsin alone decreased D2V permissiveness. The enhancement of infection by type‐specific neutralizing monoclonal antibody suggests that the D2V particles possess at least two antigenic determinants closely associated with virus infectivity.

Antibody-mediated enhancement of dengue virus infection in mouse macrophage cell lines, Mk1 and Mm1.

Abstract Antibody-mediated enhancement of dengue type 2 virus (D2V) replication in murine macrophage cell lines (Mk1 and Mm1) was studied. While both Mk1 and Mm1 supported D2V replication in the absence of enhancing antibodies, virus production was enhanced when both cell lines were inoculated with D2V in the presence of dengue type 1 virus (D1V)-hyperimmune rabbit IgG, D1V-hyperimmune mouse ascitic fluids, or D2V-hyperimmune mouse ascitic fluids at subneutralizing concentrations. The enhancement ratios were greater in Mk1 than in Mm1. Type-specific neutralizing monoclonal anti-D2V antibody also mediated D2V replication enhancement in Mk1 to the same extent as mediated by three other enhancing antibodies described above. In contrast, however, the same monoclonal antibody mediated only a slight and smaller magnitude of D2V replication enhancement in Mm1 than did the other enhancing antibodies. Fluorescent antibody observations revealed that virus replication enhancement in both Mk1 and Mm1 was due primarily to an increase in the numbers of virus-infected cells. D2V infection enhancement in Mk1 by the anti-D2V mouse ascitic fluids at a dilution showing nearly 50% plaque-reduction activity was markedly suppressed by addition of complement to the inocula, whereas that by the monoclonal antibody, which has been identified as mouse IgGl, was not. Phagocytoses of tritiated thymidine-labeled bacteria by Mk1 and Mm1 were also enhanced when the bacteria had been opsonized with antibody. The phagocytosis enhancement ratios were again greater in Mk1 than in Mm1.

Dengue Virus Plaque Formation on Microplate Cultures and Its Application to Virus Neutralization

Discussion and Summary Dengue virus plaque formation on BHK-21 cell micro-plate cultures was described. The clear plaques were visible usually 5 days after incubation in a CO2 incubator at 37°. The cells cultured in a 3-oz bottle were sufficient to prepare two microculture plates which were usually ready for use after 1-2 days of cultivation in the CO2 incubator at 37°. The overall procedures were easy and of economic advantage. It is to be stated in this connection that the affinity of dengue viruses to tissue culture cells is not necessarily high, so that the cell culture systems suitable for dengue virus plaquing have so far been limited; and even in such suitable systems the formation of clear plaques takes a much longer time of incubation than for other kinds of arbo-viruses in general. Some of the difficulties regarding this matter have been overcome by the techniques reported here. By use of the microplate cultures, in combination with the transfer-plates reported by previous investigators (8), dengue neutralization tests were performed. Technical specifics, such as dilution of serum, transfer of virus-serum mixtures, etc., could be defined with good reproducibility. A sigmoid curve relationship was revealed between the decrease in virus titer and reciprocals of antiserum concentration. Within certain grades of the serum dilution, the same relationship was linear. This is in general accordance with data obtained by other investigators (10) dealing with dengue virus plaques formed in bottle cultures. In our experiments in which the NT titers of particular serum samples measured by this method were compared with the HI titers of the same samples determined by the standard method (9), both values paralleled well with each other, indicating the compatibility of the former with the latter.

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

Localization of Dengue Virus in Nude Mice

Dengue virus (DV) is known to replicate in the brain tissue of mice when injected intracerebrally. However, other organs and tissues do not easily support the replication of DV. We have reported that a highly mouse-adapted strain of DV caused acute lethal infection in mice even when injected intraperitoneally (ip), and that host responses to DV infection, i.e., antibody production and mononuclear cell infiltration in the infected brain, were different in athymic nude (nul nu) mice and in their heterozygous littermates (nu/+) (6). Viral replication was detected not only in the brain but also in other organs and tissues of both kinds of mice. In this paper we present additional data, particularly on the morphological aspects such as localization of DV in the infected mice. DV type 1, Mochizuki strain (7), in the form of the supernatant of suckling mouse brain homogenates, was used. Four to six-week-old male Balbjc-nu/nu and nul+ mice were procured from Japan Clea Co., Ltd. Each mouse was inoculated ip with 107•0 to 107•8 plaque-forming units (PFU) of DV. At fixed times thereafter, each mouse was bled and the organs were removed. The organs were then divided into two or more portions, which were used for virus titration and histological examination, including hematoxylin-eosin (HE), periodic acid-Schiff (PAS), and indirect fluorescent antibody (FA) stains as well as electron microscopic (EM) observation. Figure I shows the growth curve and concentration of virus in the brain, skeletal muscle and heart of infected nu/nu mice. The virus was first clearly detected 4 days after infection, increased in amount and reached maximum titers at the time paralysis appeared. In the paralyzed mice, the highest virus titers were found in the brain (ca. 108 PFUIg), and the second highest in the skeletal muscle and heart (ca. 104 PFUIg). Our previous findings that the virus concentration in the heart of nu/nu mice was higher than in nul+ mice (6) were repeatedly confirmed. Lymph node, lung, liver, spleen and kidney also contained virus, although the titers varied from one sample to another. In our previous experiments it was not certain whether the virus content in the lymph node and spleen were of the same level or not, but the present studies clearly indicate that the infected lymph node had a higher concentration of virus than the spleen (Fig. 2).

Purification of arboviruses grown in tissue culture.

Discussion and Summary Arboviruses (Chikungunya, dengue type 1, and Japanese encephalitis) cultivated in monolayer cultures of BHK-21 cells grown with Eagles MEM supplemented with bovine serum albumin were purified by the following procedures: The viruses were precipitated from infectious tissue culture fluids by zinc acetate (0.05 M) and were resuspended in saturated ethylenediaminetetraacetate. The suspension was filtered through a Sephadex G200 [(lower) 10 cm]-Sepharose 6B [(upper) 40 cm] column (2.5 cm in diam) by upward elution. The filtrate was concentrated in a collodion bag in vacuo. The final step was sucrose density gradient centrifugation. The procedures were comparatively short and straightforward, and about 100 to 500 times purification factors were obtained consistently. The CHIK virus preparation after the sucrose density gradient centrifugation was demonstrated as a single peak, the infectivity and HA activity coinciding with each other; whereas the DEN-1 and JE viruses were shown to be heterogeneous. The problems of whether such a difference reflects some basic characteristics of groups A and B viruses need further investigation. Electron microscopic pictures of the preparations revealed spherical particles of (mμ): 50 to 60 (CHIK), 50 to 55 (DEN-1), and 45 to 50 (JE). These images are compatible, in general, with those previously reported by Igarashi et al. (CHIK virus) (13, 14); Matsumura and Hotta (DEN-1 virus) (15); Smith et al. (DEN-2 virus) (16); Takaku et al. (JE virus) (17); and Nozima et al. (JE virus) (18); although the origin of the viruses and the purification methods are not necessarily the same. The present purification procedures can probably be applied with success to other arboviruses in general. Further studies on the biochemical and biophysical nature of the purified virions are underway and will be reported later.

Studies on Structural Proteins of Chikungunya Virus

Chikungunya virus (CHIKV) was purified and subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in discontinuous buffer systems. Three bands were revealed by staining with Coomassie blue; two of them (E1 and E2) were associated with the membrane, and one (C) with the core. Their molecular weights were estimated to be 56,000 (E1), 52,000 (E2), and 36,000 (C), irrespective of the concentration of acrylamide in the gels. The molar ratios of Ei, E2, and C were almost equal when the sample buffer was Tris‐HCl, whereas they were different when phosphate buffer was used.

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).