Detection of a Virus Enhancing Factor in the Spheroid, Spindle, and Virion of an Entomopoxvirus Arman Wijonarko and Tosihiko Hukuhara 1 Faculty of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan Received September 25, 1997; accepted January 14, 1998 Spheroids, spindles, and virions of an entomopoxvi- rus (EPV) enhanced the infectivity of a nuclear polyhe- drosis virus (NPV) when they were perorally adminis- tered to larvae of the armyworm, Pseudaletia separata. Spheroids and spindles at the same dose exhibited nearly the same enhancing activity. When the dose of spheroids or spindles was reduced 10 times, the me- dian infectious dose of the NPV was increased approxi- mately 100 times. An antiserum against an enhancing factor detected the homologous antigen in spheroids, spindles, and tissue-derived EPV virions but not in spheroid-derived virions.  1998 Academic Press INTRODUCTION Entomopoxvirus (EPV) infection is characterized by the formation of two types of cytoplasmic inclusions: spheroid and spindle. The spheroid contains many virions within a matrix protein called spheroidin, while the spindle is composed of a protein called fusolin and contains no virions (see Arif, 1995). Croizier and Veyrunes (1971) concluded that the spindle was composed of a protein antigenically dis- tinct from the elements constituting virions on the basis of their observation that spheroid-derived virions of Melolontha melolontha EPV produced no precipitin line when reacted with an antiserum against solubi- lized spindle in immunodiffusion tests. The positive reaction of solubilized spindle to an antiserum against solubilized spheroid was ascribed by them to the pres- ence of contaminating spindles in the ‘‘spheroid’’ prepa- ration. Gauthier et al. (1995) showed that antibodies against fusolin of MmEPV detected a 50-kDa protein in dissolved spheroid in Western blot analysis. Xu and Hukuhara (1992, 1994) showed that spher- oids of Pseudaletia separata EPV contained an enhanc- ing factor (EF), which increased the infectivity of an armyworm nuclear polyhedrosis virus (NPV). Huku- hara et al. (1995) detected the EF not only in virions occluded within the spheroids but also in spindles by means of immunoelectron microscopy. This observation is not in accord with the conclusion of Croizier and Veyrunes (1971). Our objectives in this study were: (1) to establish the enhancing activity of purified virions and spindles, and (2) to determine the presence of a common antigen in them using methods other than immunoelectron micros- copy. These investigations are requisite for future analyses of the role of the EF in virosis. MATERIALS AND METHODS Virus. Stocks of Pseudaletia unipuncta NPV (Huku- hara et al., 1987) and P. separata EPV (Hukuhara et al., 1990) were propagated in larvae of P. separata. Oc- cluded EPV virions were released from spheroids with the use of a reducing alkaline buffer and then purified by density-gradient centrifugation as described previ- ously (Xu and Hukuhara, 1992). Nonoccluded EPV virions were purified from a homogenate of infected tissues in 10 mM Tris-HCl buffer (pH 7.3) as follows. The homogenate was centrifuged at 2000g for 20 min to sediment the tissue debris and inclusion bodies. The supernatant was centrifuged at 45,000g for 30 min. The pellet was suspended in the same buffer, layered over a cushion of 30% sucrose solution, and centrifuged at 40,000g for 30 min. The virus-containing pellet was sonicated while being cooled in ice water and centri- fuged on a continuous sucrose density gradient (40– 65%) in the same buffer at 50,000g for 60 min. Purified materials were placed on grids covered with a Formvar membrane, negatively stained with 1% uranium ac- etate, and examined with a JEM 100C electron micro- scope at 100 kV. Purification of EPV inclusions. Inasmuch as the previously published purification methods (Bergoin et al., 1970; Xu and Hukuhara, 1992) were unsatisfactory in separating spheroids from spindles, the following procedure was developed. Infected armyworm larvae 1 Present address: College of Bioresource Sciences, Nihon Univer- sity, Kameino, Fujisawa 252-8510, Japan. JOURNAL OF INVERTEBRATE PATHOLOGY 72, 82–86 (1998) ARTICLE NO. IN984756 82 0022-2011/98 $25.00 Copyright  1998 by Academic Press All rights of reproduction in any form reserved.