Abstract
To determine the interaction of Pseudaletia unipuncta granulovirus and Pseudaletia separata entomopoxvirus, larvae of the armyworm, P. separata, were perorally administered graded doses of entomopoxvirus occlusion bodies, spheroids, in the presence or absence of granulovirus occlusion bodies, capsules. The presence of capsules in the inocula enhanced entomopoxvirus infection. In doubly infected larvae, the inclusion bodies and virions of the two viruses coexisted in the same fatbody but never occurred in the same cells. The granulovirus appeared to facilitate entomopoxvirus infection at the organismal level, although the two viruses interfered with each other at the cellular level.
INTRODUCTION
The interactions of unrelated viruses in a common host can be examined at the cellular, organismal or ecological level in comparison with single infections, e.g., the influence of one virus on the replication of the other. There have been several laboratory investigations on the multiple interactions between a nucleopolyhedrovirus (NPV) and a granulovirus (GV) or between an NPV and an entomopoxvirus (EV) in test populations of lepidopterous larvae. When larvae were doubly infected with an NPV and a GV, interference between the two viruses was observed in Choristoneura fumiferana (Bird, 1959) and in Heliothis armigera (Whitlock, 1977) , while neither interference nor enhancement was observed in Trichoplusia ni (Lowe and Paschke, 1968) , in Hyphantria cunea (Hukuhara et al., 1969) and in Adoxophyes orana (Fluckiger, 1982) . Tanada (1956 ) demonstrated a synergistic association between an NPV and a GV in Pseudaletia unipuncta. Similar findings were reported for NPV-GV combination studies with P. separata (Hukuhara et al., 1987) , T. ni (Derksen and Granados, 1988) and Xestia c-nigrum (Goto, 1990) . Xu and Hukuhara (1992) and Mitsuhashi et al. (1998) demonstrated NPV-EV synergistic associations in P. separata and Bombyx mori, respectively.
INTRODUCTION
The present study was initiated to determine the interaction between two NPV synergists, GV and EV, in P. separata. This is the first report of a synergistic interaction between the two viruses.
MATERIALS AND METHODS
The Hawaiian strain of P. unipuncta GV (Psun-GV) (Tanada and Hukuhara, 1968 ) and a Chinese isolate of P. separata EV (PSEV) (Hukuhara et al., 1990) were propagated in the armyworm, P. separata, by administering perorally the virus stocks to fourth-instar larvae. The inclusion bodies of the two viruses (capsules, spheroids, spindles) were purified from heavily infected larvae, as described previously (Yamamoto and Tanada, 1978; Xu and Hukuhara, 1992) and suspended in distilled water. The PSEV inclusion bodies in the resultant suspensions consisted of 90% spheroids and 10% spindles. Their concentrations were determined using a hematocyte-counting chamber (Thoma type, Erma, Tokyo). The concentrations of the capsules in the PsunGV suspensions were determined electron-microscopically, as described previously (YoboeiseiKenkyusho-Gakuyukai, 1964; Hukuhara et al., 1987) . The inclusion body suspensions of the two viruses were kept at 5°C until used.
MATERIALS AND METHODS
To prepare virus inocula, four different concentrations of a PSEV suspension were combined with Enhanced infection of an entomopoxvirus in larvae of the armyworm, Pseudaletia separata (Lepidoptera: Noctuidae), by a granulovirus (Hattori and Atsusawa, 1980) in Erlenmeyer flasks. Larvae soon after ecdysis were transferred to a non-aseptic condition and starved overnight. They were individually placed in a plastic vial (6.0 cm in diameter, 3.5 cm in height) and administered a piece (0.1 g) of artificial diet that had been covered with 20 ml of virus suspension. Within two days, fresh virus-free diet was administered to larvae that had consumed the virus-contaminated diet. The test larvae were observed daily until pupation. Diseased or dead individuals that occurred during this observation period (about two weeks) were diagnosed by light-microscopic examination of a piece of the fat body for the presence of inclusion bodies (GV capsules, EV spheroids and spindles) characteristic of the two virus infections. For electron microscopy, fat bodies from doubly infected larvae were fixed in 2.5% glutaraldehyde in cacodylate buffer (pH 7.4), postfixed for 1 h in 1% osmium tetroxide in the same buffer, dehydrated in ascending grades of ethanol and acetone, and embedded in Epok 812 (Epoxy Equivalent 148, Oken Shoji, Tokyo). Ultrathin sections were stained with uranyl acetate and lead salts (Sato, 1970) and viewed with a JEM-1200EX electron microscope at 80 kV.
MATERIALS AND METHODS
The median infectious doses (ID 50 s) and their standard errors were calculated according to Berkson (1955) . The enhancing effect was determined by computing the ratio between the ID 50 of a PSEV dilution series combined with capsules and the ID 50 of the same PSEV dilution series combined with distilled water. The ratio was converted to a log 10 value and referred to as the enhancement index log 10 .
RESULTS AND DISCUSSION
In the first set of bioassays, armyworm larvae were administered four graded doses of EV inclusion bodies ͗spheroids and spindles͘ which had been combined either with a constant dose of GV capsules or distilled water. Simultaneous administration of capsules at 3.6ϫ10 9 /larva and spheroids at doses of 1.9ϫ10 2.5 -1.9ϫ10 4.0 /larva resulted in larvae that were infected with either EV alone (3-44%), GV alone (0-63%) or both viruses (8-46%) (Fig. 1) . The sum of the percentage of EV infection alone and that of double virus infection increased directly as the EV dose was increased. When only EV inclusion bodies were administered, 10% and 3% of the test larvae were EV-infected at the highest (1.9ϫ10 4.5 spheroids/larva) and the second highest (1.9ϫ10 4.0 spheroids/larva) doses, respectively. No virus infection occurred in larvae that had been administered two lower spheroid doses (1.9ϫ10 3.0 , 1.9ϫ10 3.5 /larva) or in control larvae that had been administered diet moistened with distilled water. Administration of capsules alone at 3.6ϫ10 9 /larva gave rise to GV infection in 76% of the test larvae.
RESULTS AND DISCUSSION
In the presence of capsules, the EV ID 50 was 2.2ϫ10 3 spheroids/larva and the 95% fiducial limit of the log 10 ID 50 was 3.19-3.47. The ID 50 in the absence of capsules was estimated to be greater than the highest dose (1.9ϫ10 4.5 spheroids/larva) tested, although this cannot be determined with certainty because of the absence of infected larvae in the lower two doses. Inasmuch as this estimated value is greater than the lower limit of the ID 50 in the presence of capsules, it is presumed that the GV enhanced EV infection.
RESULTS AND DISCUSSION
In the next set of bioassays, a wider range of spheroid doses (2.5ϫ10 2.0 -5.0ϫ10 5.0 /larva) were tested in the presence, and in the absence, of a 256 T. HUKUHARA et al. Fig. 1 . Histograms representing the total percentages of armyworm larvae with virus infections. There were 40 larvae per dose. The larvae were administered EV suspension alone (A) or EV suspension combined with GV suspension (B) . No virus infection occurred in 40 control larvae. reduced dose (2.1ϫ10 9 /larva) of capsules (Fig. 2) . The percentage of larvae with EV infection increased directly as the spheroid dose was increased in both dilution series. Simultaneous administration of GV and EV suspension resulted in larvae infected with either EV alone (10-37%), GV alone (7-17%) or both viruses (7-37%). Administration of capsules (2.1ϫ10 9 /larva) alone resulted in GV infection in 20% of the test larvae. No virus infection occurred in control larvae.
RESULTS AND DISCUSSION
The EV ID 50 s were 8.7ϫ10 3 spheroids/larva in the presence of capsules and 2.3ϫ10 5 spheroids/ larva in their absence ( Table 1 ). The 95% fiducial limits of the two log 10 ID 50 s were broad, but did not overlap, indicating a statistically significant difference. The enhancement index log 10 was 1.43, which meant that EV infection was enhanced 27-fold.
RESULTS AND DISCUSSION
At the advanced stage of infection, both GV-and EV-infected larvae became yellowish in comparison with the light-brown color of healthy larvae due to the presence of a large number of inclusion bodies in the fat body, which could be seen through the semi-transparent larval integument. Diagnosis of double infection was based on the coexistence of GV inclusion bodies (capsules) and EV inclusion bodies (spheroids and spindles) in the same larva. GV-infected fat body cells appeared brownish due to the massive accumulation of capsules in the cytoplasm. EV-infected cells contained numerous inclusion bodies (spheroids, spindles) but the change in cell color was not observed. Examination of the fat body from more than 70 doubly infected larvae revealed that GV-infected cells occurred less frequently than EV-infected cells and that GV and EV inclusion bodies never occurred in the same cells. Electron-microscopic examination of ultrathin sections of doubly infected fat bodies showed that not only the inclusion bodies but also the virions of the two viruses occurred in separate cells (Fig. 3) . The results indicated that the two viruses interfered with each other at the cellular level.
RESULTS AND DISCUSSION
We have shown that the GV enhances the EV infection at the organismal level. The capsule of the Hawaiian strain of PSEV contains a factor, called synergistic factor or PsunGV enhancin, that facilitates NPV infection in armyworm larvae (Tanada and Hukuhara, 1971; Hukuhara et al., 1987; Roelvink et al., 1995) . It is tempting to speculate that the synergistic factor also facilitates EV infection.
RESULTS AND DISCUSSION
The practical implications of multiple virus interaction are dependent on the combination of viruses and the specific host in question. In microbial control of insect pests, the combination of two viruses that interact by synergism may purposely be used to enhance the effect of a viral insecticide formulation. Hukuhara et al. (1999) have suggested the use of a virus enhancing factor from PSEV as an additive of NPV insecticides. For the opposite purpose, that is to say, to protect useful insects from viral infections, the knowledge of multiple virus interactions should also be useful. Aruga et al. (1965) silkworm larvae with the flacherie virus can prevent the occurrence of the cytopolyhedrovirus when this virus is subsequently administered. If the flacherie virus is rendered non-pathogenic, it may be purposely administered to silkworm larvae to protect them from cytopolyhedrovirus infection. The modulation of viral infection by viral interactions may assume a more significant role in insects than in vertebrates, which generally respond by active production of specific antibodies directed against the viruses. Fig. 3 . Ultrathin section of a fat body from a doubly infected armyworm larva. Note the presence of occlusion bodies and virions of Pseudaletia unipuncta granulovirus in one cell and those of P. separata entomopoxvirus in the adjoining cell. GV, cell infected with the granulovirus; EV, cell infected with the entomopoxvirus; C, capsule; S, spheroid. Arrows point to virions of the two viruses. Barϭ1 mm.

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