Tosihiko Hukuhara

Enhanced infection of a nuclear polyhedrosis virus in larvae of the armyworm, Pseudaletia separata, by a factor in the spheroids of an entomopoxvirus

Abstract Spheroids of an entomopoxvirus (EPV) enhanced the infectivity of a nuclear polyhedrosis virus (NPV) for larvae of the armyworm, Pseudaletia separata. When larvae were perorally administered a mixture of the spheroids and polyhedra, there was a direct increase in the number of larvae infected with the NPV as the dose of the spheroids increased. A proteinaceous factor within the spheroid was responsible for the enhancement. The factor retained its enhancing activity when dissolved with a mixture of Na2CO3, sodium thioglycolate and EDTA. The supernatant fraction of solubilized spheroids, which had been obtained by centrifugation at 48,000g, was more active than the pellet fraction in enhancing the NPV infectivity. Further fractionation of the supernatant on a sephacryl column revealed that the enhancing activity was present in one of the three proteinaceous peaks. The factor was 38 kDa in molecular mass and serologically unrelated to a synergistic factor of a P. unipuncta granulosis virus in double-diffusion tests. The replication of the EPV was hampered by the NPV in multiple infections.

Replication of an entomopoxvirus in two lepidopteran cell lines

Pseudaletia separata entomopoxvirus replicated in two lepidopteran cell lines, SIE-MSH-805-F and BM-N. Microscopic examination, and the virus passage tests, of infected cultures indicated that the virus replicated more readily in the former cell line. Virus release by exocytosis occurred in both cell lines. A sequence of virus morphogenesis in the cultured cells was described, based on electron microscopic observations of thin sections. The nucleus of infected cells contained spherical inclusions, and the cytoplasm contained virions, immature virus forms, spheroids, and spindles. A portion of the virions in the cytoplasm was occluded within spheroids, which were often associated with crystallogenic matrix. Virions acquired a coat prior to their occlusion.

Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta.

A synergistic factor (SyF), which enhanced the infection of nuclear polyhedrosis viruses, was purified from capsules of a Pseudaletia unipuncta granulosis virus (Hawaiian strain) by immune affinity chromatography. The isolated SyF consisted primarily of a protein with molecular mass 98 kDa. The recovery rate depended on the alkali used to dissolve the capsules: the highest rate occurred with 0.05 M Na2CO3-0.05 M NaCl, followed in turn with 0.02-0.05 M NaOH and 0.04 M NaOH-0.05 M glycine. The solubilized components from untreated capsules contained 98- and 100-kDa proteins in addition to the matrix protein (29 kDa) and its decomposed products, while those from heat-treated capsules contained only the 100-kDa protein. Virons liberated from the capsules with the glycine buffer contained three proteins (33, 98, and 100 kDa) serologically related to the SyF. Immunoelectron microscopy of infected tissue and purified virions revealed the localization of the SyF antigens on the viral envelope.

Enhancement of the in vitro infectivity of a nuclear polyhedrosis virus by a factor in the capsule of a granulosis virus

Abstract A synergistic factor (SyF) present in the capsules of the Hawaiian strain of Pseudaletia unipuncta granulosis virus enhanced the infection of Spodopera frugiperda cells (IPLB-SF21AE) in culture with plasma-membrane-budded and polyhedra-derived virions of Trichoplusia ni multinucleocapsid nuclear polyhedrosis virus (TnNPV). Bombyx mori NPV, P. unipuncta NPV, and TnNPV, when assayed in their homologous cell lines in the presence of the SyF, were not enhanced in their infectivity. Specific binding of the SyF to the surface of S. frugiperda cells and to the envelopes of TnNPV and P. unipuncta NPV was visualized by immunofluorescence microscopy and immunoelectron microscopy, respectively. The SyF had no specific affinity to cell lines from B. mori (BM-N), P. separata (NIAS-LeSe-11), and T. ni (TN-368) or to polyhedra-derived virions of B. mori NPV. Anti-SyF antibodies and chloroquine inhibited the activity of the SyF.

Rhythmic contractile movements of the larval midgut of the silkworm, Bombyx mori

Abstract The raidgut of intact larvae of Bombyx mori exhibited active contractile movements. Contraction waves were generated rhythmically at several regions of the midgut. The waves passed in both oral and aboral directions from their sites of origin. Midgut movements were depressed during moulting. The midgut continued to move normally when tetrodotoxin was injected into the larval haemocoel at doses sufficient to paralyze somatic muscle. Ligation of larvae paralyzed with tetrodotoxin behind the second or third body segment resulted in the abolition of the contraction waves in the midgut portion anterior to the ligature. A ligature applied behind other body segments did not hamper midgut motor activity irrespective of whether or not an abdominal ganglion had been extirpated. The frequency of contraction and the rate of food transport in the midgut were increased when larvae were administered serotonin or when their body temperature was raised.

Purification of polyhedra of a cytoplasmic polyhedrosis virus from soil using metrizamide

T. Hukuhara (J. Invertebr. Pathol., 25, 337-342, 1975) developed a method of purifying virus polyhedra from the soil based on the following three steps: (1) desorption of polyhedra with sodium pyrophosphate; (2) aqueous two-phase separation; and (3) CsCl density-gra~ent cen~ifugation. The recovery of polyhedra by this method was low because of incomplete desorption in step 1 and soil aggregation caused by the high ionicity in step 3. If the method is used for field application, large amounts of soil will be needed because the soil is likely to contain only a relatively small amount of virus, and the sample must be concentrated prior to step 3 to reduce the sample volume to accommodate for high-speed centrifugation. We have investigated the effect of ultrasonication in step 1, the procedures for concentrating the sample obtained in step 2, and the use of a nonionic gradient material in step 3. Polyhedra of a cytoplasmic polyhedrosis virus of Bombyx mori were thoroughly mixed with brown forest soil (log polyhedra/g of soil). The preparation of the mixture, the desorption of polyhedra, and the separation by a biphasic system were performed as in the original method, except that some samples were ultrasonicated for 1 hr at 30 kc (700 W) in an ultrasonic oscillator (Toyo Riko Seisakusho, Tokyo, Type UD-N-50-6) before being incorporated into the biphasic system (Table 1). Contrary to expectation, both the recovery rate of polyhedra and the purification factor were lower when the samples were ultrasonicated (Table 2). The ultrasonicated samples contained a higher proportion of clay particles because of the decomposition of the larger soil particles. This resulted in a ninefold increase in the amount of soil entering the upper phase of the biphasic system. The reduced recovery rate of polyhedra was presumably due to the increased soil surface which adsorbed the polyhedra. After biphasic separation, the upper phase was diluted with an equal volume of distilled water and was centrifuged for 1 hr at 1800 rpm (65Og) to sediment the soil and polyhedra. The pellet was resuspended in a small volume of distilled water, about one-eighteenth of the original volume of the upper phase and was thoroughly mixed with an equal volume of an 80% (w/v) solution of metrizamide [2-(3-acetamido-5-N-methylacetamido 2,4,6 triiodobenzamido) 2 deoxy-D-glucose] (Nyegaard & Co. A/S, Oslo, Norway) in 0.2 M Tris buffer,pH 7.0. Some samples were combined with a CsCl solution as in the original method and served as controls. After centrifugation of the nonultrasonicated samples for 20 hr at 60,000 rpm (314,000g at R,,,) in a Beckman fixedangle rotor (Type 65Y), a sharp white band of polyhedra appeared in the middle of the metrizamide density-gradient column and at the top of the CsCl column. When ultrasonicated samples were used, the bands were more diffuse, and the number of polyhedra recovered was less in the metrizamide column but nearly the same in the CsCl column. The best recovery of purified polyhedra was obtained when the nonultrasonicated samples were centrifuged in the metrizamide column (Table 2). We tested two other biphasic systems for their efficiencies in concentrating the sample prior to step 3, as compared to the simple centrifugation described above. When the upper phase obtained in step 2 was mixed with l/100 vol of 20% dextran sulfate 500 (Pharmacia, Uppsala, Sweden) and l/l0 vol of 3 M NaCl and was placed overnight in the cold, the mixture separated into two phases, an upper polyethylene

Structure of aggregates of Tipula iridescent virus and polystyrene latex particles

Abstract Tipula iridescent virus aggregated with polystyrene latex particles 126 nm in diameter. There was a region of optimal proportion of the two particles. The aggregation proceeded faster and the amount of resultant aggregates was larger at the higher concentrations of the two particles, but the size of the individual aggregates was smaller. The aggregates consisted of clusters of the two particles with vacant spaces interspersed among them. A hypothetical model of the particle arrangements was presented. The aggregates were reversibly dissolved in 0.03% sodium dodecyl sulfate and 30% n -propanol and isopropanol. In the presence of lower concentrations of these solvents, the aggregation occurred at high temperatures but not at low temperatures. These results were interpreted as implicating hydrophobic interactions in the formation and stability of the aggregates.