INTRODUCTION

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Persistent/latent viral infection is a long-term association between a host cell and a virus. It has been recognized to occur in insects since the last century, and depending upon external conditions, this chronic course will often change suddenly into an active or acute infection (36). Today, although viral persistence has been speculated to occur with many different viruses, little is known about the associations between these viruses and their hosts during their long quiescent periods (9, 25, 36). In this study, Hz-1 virus (also termed Hz-1 baculovirus, Hz-1V, and HzV-1) was used to elucidate the association and physical status of a persistent viral infection in insect host cells. Hz-1 virus was formerly recognized as the type species of the subfamily Nudibaculoviridae of the family Baculoviridae (43). More recently it and other nonoccluded baculoviruses have been removed from the baculovirus family, and they are currently unclassified (41).

The long-term association between Hz-1 virus and host cells is generally referred to as persistent viral infection. Hz-1 virus was identified by Granados and coworkers in 1978 (22) as a persistent infecting baculovirus agent in the IMC-Hz-1 ovarian cell line. It is an enveloped, nonoccluded, rod-shaped virus that contains a double-stranded circular 228-kb DNA genome (12, 24). The virus particles are usually heterogeneous in length according to examination by electron microscopy. Following plaque purification under conditions of productive infections, virus particles that are homogeneous in length can be purified (8, 12).

Electron microscopic examination of the purified homogeneous virus particles (referred to as standard virus) indicates that the mean particle length is 414 nm. However, after several serial, high-multiplicity passages of standard virus, the amount of defective interfering particles increases significantly (8). The length of the defective interfering particles is variable, and they are generally shorter than standard particles. This may be due to deletions in the standard viral genome (8, 12). Later, it was found that persistently infected cell lines could be established from the few viable cells remaining after acute infections (9, 12, 13, 28). Subcultures of the established persistently infected cell lines may release infectious viruses into the medium (9, 32, 37). Nevertheless, since cells persistently infected with Hz-1 virus look normal and grow well, it is not known if virus particles are released from a small portion of cells that were spontaneously reactivated or from all cells but with relatively low virus yields per cell (44).

The host range of Hz-1 virus is broad. Insect cell lines from seven lepidopterans, including Trichoplusia ni (TN368), Spodoptera frugiperda (IPLB-SF-212), Heliothis zea (IPLB-1075), Mamestra brassicae, Porthetria dispar (IPLB-65Z), Lymantria dispar (LD252Y), and Heliothis virescens (BCIRL-HB-AM1) are susceptible to Hz-1 virus infection (44). Persistent Hz-1 virus infections have been established in three of these insect lines: H. zea, T. ni, and S. frugiperda (12, 13, 22, 44). Once host cells are persistently infected with Hz-1 virus, they are resistant to superinfection with the same virus (9), and such homologous interference occurs simultaneously with the induction of cellular apoptosis upon viral challenge (28). Further experiments showed that apoptosis can be blocked by the p35 gene (16) derived from Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) and that homologous viral interference results either from a mechanism upstream from the function of the p35 gene or from a mechanism other than apoptosis induction (29).

Previously, differential Hz-1 viral transcriptions under conditions of productive and persistent infections were reported. During productive viral infections, more than 100 transcripts are detected, whereas only a single viral transcript, the persistence-associated transcript 1 (PAT1), has been detected during persistent viral infections (13). This provides an opportunity for the study of the mechanisms of differential viral gene expression during both viral infection cycles in insect cells. Currently, the physical status and the associations of the Hz-1 virus with persistently infected insect cells are still largely unknown. In this study, we have developed a novel technique to study the rate and status of viral reactivation. We have also applied pulsed-field gel electrophoresis (PFGE) and in situ hybridization techniques to analyze the physical status of viral genomes in persistently infected cells. Our results clearly show that some of the viral genomes do insert into the host chromosome, and this suggests that persistent Hz-1 viral infection should be more accurately referred to as latent viral infection.

	MATERIALS AND METHODS

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Cells and viruses. The T. ni (TN368) and S. frugiperda (SF21AE and SF9) cell lines were maintained at 26°C in a modified TNM-FH medium as described by Burand et al. (8) and Chao et al. (12). Standard Hz-1 virus was derived by plaque purification with SF21 cells (8). Persistently infected cell lines TNP1, TNP2, TNP3, SFP2, and SFP4 were derived from serial high-multiplicity (multiplicity of infection [MOI] = 10) passages of the standard virus by the procedures of Burand and Wood (10). The TNP1, TNP2, and TNP3 cell lines were derived from TN368 cells, and the SFP2 and SFP4 cell lines were derived from SF21AE and SF9 cells, respectively.

Assays of viral release from fluorescence-labeled persistently infected cells. The cell membranes of persistently infected cells were first labeled with CellTracker CM-DiI (Molecular Probes). CellTracker CM-DiI is a fluorescent carbocyanine dye which specifically stains the membranes of cells without obvious diffusion to the medium or other cells, as reported by the manufacturer's instruction manual. We found that the fluorescent dye persists on insect cells for at least 5 days without any adverse effects on cell growth or on the process of reactivation of persistently infected viruses (data not shown).

Southern and Northern analyses. Total cellular and viral DNAs were purified as previously described (12, 13). Purified DNAs were digested with restriction enzyme EcoRI and fractionated by electrophoresis through a 0.8% agarose gel for 44 h at 3 V/cm. After ethidium bromide staining and photography under short-wavelength illumination, the DNA was transferred to a GeneScreen filter (Dupont), hybridized with ³²P-random-primer-labeled standard viral DNA, and autoradiographed. Northern hybridizations were performed as described previously (13). Briefly, samples containing 3 µg of total RNAs extracted from healthy and persistently infected cells were treated with glyoxal and fractionated through a 1% agarose gel. After blotting, the filter was hybridized with a ³²P-random-primer-labeled probe prepared from subfragment D of the viral EcoRI-M fragment (13). For these experiments all of the persistently infected cells were assayed at passages 200 to 230, except SFP4 cells were used at passage 30.

Slot hybridizations. Total genomic DNAs were purified from healthy (TN368 and SF21AE) and persistently infected (TNP1, TNP2, TNP3, and SFP2) cell lines and transferred to the GeneScreen filter by using MilliBlot (Millipore) according to the manufacturer's instructions. The filter was hybridized with the ³²P-labeled standard viral DNA, followed by autoradiography.

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PFGE analyses. Healthy or persistently infected TN368 and SF cell lines (2 × 10⁷ cells) were mixed with equal volumes of 1% low-melting-point agarose prepared in phosphate-buffered saline (PBS) and cooled to 45°C. These samples were then transferred to plug molds with a pipette and allowed to harden at 4°C. Sample blocks were transferred to Eppendorf tubes which contained 3 to 5 volumes of 0.5 M EDTA (pH 9)-1% sarcosyl-0.5 mg of proteinase K per ml. These blocks were digested for 1 to 2 days at 50°C with constant, gentle shaking. After digestion with SmaI or NotI restriction enzyme, PFGE was carried out with a CHEF-DRII pulsed-field electrophoresis system (Bio-Rad Laboratories) in a 1% agarose gel with 0.5× TBE buffer (40 mM Tris-borate, 1 mM EDTA) at 200 V with a switch time of 60 s for 15 h followed by a switch time of 90 s for 8 h at 14°C. The gel was stained in water containing 0.5 µg of ethidium bromide per ml and photographed under short-wavelength UV illumination. The gels were transferred to a GeneScreen filter (Du Pont), hybridized with ³²P-labeled standard viral DNA, and autoradiographed. In these experiments, TNP1, TNP2, and TNP3, and SFP2 cells were assayed at passages 200 to 230, and SFP4 cells were assayed at passage 30.

Chromosomal fluorescence in situ hybridization. For fluorescence in situ hybridization of viral DNA in persistently infected cells, an RNA probe was made as follows. A 0.3-kb HincII subfragment derived from HindIII-K of the viral genome (12) was cloned into plasmid pBluescript KSM(+) (Stratagene Cloning Systems), and a 0.3-kb RNA probe labeled with digoxigenin-11-UTP was produced from this plasmid by in vitro transcription with T3 RNA polymerase (Boehringer Mannheim).

Electron microscopy. SF9 cells at 17 h postinoculation with Hz-1 virus as well as TN368 and TNP3 persistently infected cells (10⁶; at passages 200 to 230) were harvested and washed in 0.1 M sodium cacodylate buffer (pH 7.2). The cell pellets were fixed for 1 h with a mixture of 4% paraformaldehyde and 0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C and then postfixed for 1 h with buffered 1% osmium tetroxide at 4°C. After dehydration in a sequential ethanol series, the cells were infiltrated and embedded in LR white. Ultrathin sections were stained for 30 min in uranyl acetate and for 5 min in lead citrate. Grids were examined with an EM902 (Zeiss) electron microscope. Productively infected SF9 cells were also harvested at 17 h after viral infection (MOI = 5) and treated by the same procedure described above to show the size and morphology of viral particles under the same electron microscopic conditions.

	RESULTS

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Mature, infectious viruses are produced in only a small proportion of low-passage, persistently infected cells. When five low-passage (passage 30) persistently infected cell lines, TNP1, TNP2, TNP3, SFP2, and SFP4, were analyzed, infectious viruses could be detected in the media; however, no infectious viruses were detected in any of the persistently infected cell lines after passage 200 (Table 1). Although newly established persistently infected cells produced viruses when seeded onto healthy cells as described in Materials and Methods, very few plaques were produced from all lines, ranging from 21 plaques/10⁵ cells for TNP3 and SFP2 cells to 210 plaques/10⁵ cells for TNP1 cells (Table 1). These results clearly show that within 5 days, the time needed for plaque formation, fewer than 0.2% of the persistently infected cells tested produced virus. However, plaques were generated by the viruses released from a small percentage of persistently infected cell lines, suggesting that mature, infectious viruses were indeed generated from these cells.

View larger version (196K): [in this window] [in a new window]   FIG. 1.   Focus-forming assay with fluorescently labeled, persistently infected cells. Fluorescence-labeled persistently infected TNP3 cells were mixed with healthy SF21AE cells. (A and C) Photographs taken with visible light; (B and D) photographs taken with a combination of visible and 530-nm excitation lights. (A and B) Cells taken from the same region, where plaques were not observed. In these regions, both healthy lawn SF21AE cells and labeled TNP3 cells (arrowheads) grew together without any sign of viral infection. A higher magnification of the labeled TNP3 cell is shown in the inset. (C and D) Plaque generated in a lawn of SF21AE cells following the release of viral progeny from a persistently infected TNP3 cell. The TNP3 cell (arrowhead) at the center of the plaque is also shown at a higher magnification in the insets.

All persistently infected cells express PAT1 and are highly resistant to superinfection. Total RNA was extracted from persistently infected cells and fractionated by agarose gel electrophoresis. After Northern blot hybridization, the PAT1 RNA, previously reported to be the only detectable persistence-associated transcript (11, 13), was found in all persistently infected cells. PAT1 was expressed more strongly in TNP1 and SFP4 cells than in TNP2, TNP3, and SFP2 cells (Fig. 2). We found that the intensity of PAT1 expression was higher in newly established SFP4 cells (passage 30) than in long-passaged persistently infected cells (passages 200 to 230). Currently it is not known if this is because of cell line variation or because a newly established persistently infected cell line usually tends to more strongly express PAT1 (29).

View larger version (59K): [in this window] [in a new window]   FIG. 2.   Detection of PAT1 expression in persistently infected cells. Total RNA was extracted from healthy (TN368 and SF21AE), productively infected (TN368 + Hz-1), and persistently infected (TNP1, TNP2, TNP3, SFP2, and SFP4) cells. The expression of PAT1 was detected by Northern hybridization with a 32P-labeled subfragment D of the viral EcoRI-M fragment, by which PAT1 is encoded (13), as a probe.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Viral interference assay of parental and persistently infected cells. Parental and persistently infected cells were challenged with either Hz-1 virus (A) or AcMNPV (B) at an MOI of 0.1. TN368 and SF21AE cells are healthy parental cells and serve as controls for viral infection. At 72 h postinfection, the titers of the viruses released into the media by the infection in two parental and five persistently infected cell lines were assayed. Data (means ± standard deviations) were collected from three sets of experiments with three independent analyses of the 50% tissue culture infective dose (TCID50).

High DNA content and coexistence of standard viral genomes and viral genomes with deletions in persistently infected cells. The viral DNA contents of the persistently infected cell lines were determined by slot blot hybridization. Total DNA was purified from the TNP1, TNP2, TNP3, SFP2, and SFP4 cell lines. Increasing amounts of total cellular DNAs and standard viral genomic DNAs were slot blotted onto a nylon filter and hybridized with ³²P-labeled viral genomic DNA for Southern blot analysis. The results showed that the percentages of viral DNAs in TNP1, TNP2, TNP3, SFP2, and SFP4 cells were 5.3, 2.1, 16.5, 0.12, and 0.66%, respectively (Fig. 4 and Table 1).

View larger version (63K): [in this window] [in a new window]   FIG. 4.   Autoradiogram of a dot blot hybridization to calculate the percentage of viral DNA in persistently infected cells. The indicated amounts of total DNA extracted from two parental and five persistently infected cell lines were dot blotted onto filters and hybridized with 32P-labeled viral genomic DNA. The indicated amounts of Hz-1 viral DNA were also dot blotted onto filters and hybridized simultaneously to serve as standards for the calibration of viral DNA in different cell samples. The two lanes for each DNA species represent duplicate samples.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   Autoradiogram of Southern blots of viral and cellular genomic DNAs. Total genomic DNAs of parental and persistently infected cells were digested with the restriction enzyme EcoRI. After fractionation through an agarose gel, genomic DNAs were blotted onto a filter and then hybridized with a viral genomic DNA probe. Arrowheads indicate extra fragments, and asterisks indicate multimolar bands which were detected from the viral genome of persistently infected cells. Hz-1, EcoRI-digested standard viral DNA.

PFGE analysis of the physical state of the viral genome in persistently infected cells. DNA samples from viral particles and different persistently infected cells were prepared in low-gelling-temperature agarose. The DNAs were separately incubated with and without two restriction enzymes, SmaI (for which Hz-1 viral DNA has only one recognition site) and NotI (for which Hz-1 viral DNA has no recognition site) (12). After restriction digestion, the samples were loaded onto the gel. Based on ethidium bromide staining results, the majority of viral DNAs remained in the well prior to SmaI digestion (Figs. 6A and B, panels a, lanes 1). Following SmaI digestion, a single-species DNA of about 228 kb migrated into the gels (Fig. 6A and B, panels a, lanes 2). By Southern hybridization analysis, the viral DNA was detected primarily in the wells prior to SmaI digestion; however, a small amount of viral DNA with a size of about 228 kb was detected (Fig. 6A and B, panels b, lanes 1). This minor 228-kb viral DNA may have resulted from nicking of some of the viral genomes during sample preparation. After SmaI digestion, the majority of DNA isolated from viral particles was converted to 228-kb linear molecules (Figs. 6A and B, panels b, lanes 2). A portion of the viral DNA remained at the origin, presumably representing viral genomes whose protein coats were not completely digested by proteinase K during sample preparation and were thus trapped in the sample wells.

View larger version (48K): [in this window] [in a new window]   View larger version (30K): [in this window] [in a new window]   View larger version (46K): [in this window] [in a new window]   FIG. 6.   PFGE analysis of the physical status of viral genomes in persistently infected TN and SF cells. (A and C) Analysis of DNA isolated from persistently infected TNP1, TNP2, and TNP3 cells, TN368 control cells, and Hz-1 virions with and without SmaI (A) and NotI (C) restriction enzyme digestion. (B) Analysis of DNA isolated from persistently infected SFP2 and SFP4 cells, SF21AE control cells, and Hz-1 virions with and without SmaI restriction enzyme digestion. Panels a, ethidium bromide-stained electrophoreograms; panels b, Southern analyses of panels a probed with 32P-labeled genomic DNA of Hz-1 virus. Brackets, regions of autoradiograms containing viral DNA sequences larger than the linearized viral genome; arrowheads, positions of DNA molecules larger than 1 Mb; asterisks, positions of the linearized unit viral genome.

Chromosomal in situ hybridization analysis further reveals the existence of both chromosomally inserted and free viral DNAs in persistently infected cells. In order to further verify the physical status of the viral genomes in the nuclei of persistently infected cells, chromosomal in situ hybridization was performed. Persistently infected TNP3 cells were fluorescence in situ hybridized by using a probe derived from the HindIII-K region of the viral genome (12). The probe was labeled with fluorescein isothiocyanate-conjugated antibody, which resulted in a green color, whereas the host chromosomes were stained with propidium iodide, which resulted in a red color. When signals of the viral genome overlapped with those of host chromosomes, they turned yellow.

View larger version (40K): [in this window] [in a new window]   FIG. 7.   Genomic analysis of Hz-1 virus by in situ hybridization with persistently infected TNP3 cells. Genomic signals of Hz-1 virus were detected in interphase (nuclei 1 and 2) and mitotic (nucleus 3) cellular chromosomes. The metaphase spread was counterstained with propidium iodide to outline chromosome morphology (red). The probe derived from the viral genomic DNA was labeled with fluorescein isothiocyanate-conjugated antibody, which resulted in a green color; the overlapping of the viral signal and host chromosome produced a yellow color. (A) Original unprocessed image. (B to E) Series of digitized images of the host chromosomes obtained by optical sectioning (z-axis spacing = 1 µm). Arrows indicate those viral signals which were detectable only in middle sections (C and D) and were thus embedded inside the host chromosomes. (F) In situ hybridization of interphase and mitotic chromosomes of healthy TN368 cells with the same viral probe as negative controls. (G and H) In situ hybridization of dividing TNP3 nuclei, showing equal viral DNA signals associated with the newly divided daughter chromosomes. (G) Viral signals only; (H) dual images of viral signals and propidium iodide-stained nuclei.

View larger version (166K): [in this window] [in a new window]   FIG. 8.   Electron microscopic view of cells productively and persistently infected with Hz-1 virus. (A) Uninfected TN368 cell. (B) SF9 cell productively infected with Hz-1 virus. Virus particles (V) are indicated with arrowheads. (C and D) Persistently infected TNP3 cells with no detectable virions. Nm, nuclear membrane; N, nucleus; Cy, cytoplasm; Mt, mitochondria. Bars, 2.5 µm.

	DISCUSSION

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In this study we demonstrated that relatively newly established cell lines persistently infected with Hz-1 virus produced virus, whereas the production of virus ceased after 200 passages. We do not rule out the possibility that viral reactivation may still occur after many passages due to physiological or environmental stimulation. In newly established cells persistently infected with Hz-1 virus, infectious virus was produced from a minor number of cells, and these cells died or stopped growing upon the release of viral progeny. This is a phenomenon very similar to that of latent herpesviruses.

Viral reactivation in herpesviruses is best studied by monitoring the appearance of the viral antigens associated with productive viral infection (6, 23, 33, 35). The infected cells used for experimentation are usually damaged from the fixation procedure necessary to detect viral antigens. As a result, whether the mature viruses are finally produced from particular reactivated cells and whether specific individual cells die or survive and become virus producers after reactivation can be elucidated only for populations of cells, not for individual cells (5, 42). By taking advantage of a viable long-term membrane-specific fluorescent dye, we designed a simple and reproducible plaque assay technique to verify the nature of viral reactivation and to examine the final fate of the cells in which virus is reactivated. It was found that during early stages of persistent infection, viral progeny were generated from only very small proportions of host cells, which is comparable to what occurs with herpesviruses (42).

Because the copy numbers of the Hz-1 viral genome are unusually high and because the genomes contain complicated genomic deletions or insertions (Fig. 5) (12), the physical status of viral DNA in persistently infected cells is an interesting yet difficult issue to study by conventional molecular biological techniques. In order to resolve these problems, PFGE techniques were applied. It has been reported that under the conditions of PFGE, circular DNA is trapped at the top of the gel; however, linear DNA is amenable to gel separation (30, 39). This explains why in the present analysis of virus particles, the intact viral DNA was trapped at the top of the gel but migrated to about the 228-kb region after SmaI digestion. Similar phenomena were also observed for the digestion of total genomic DNAs of various persistently infected cells, suggesting that circular viral DNAs may also exist in these cells. Our results show that viral DNA is inserted into the genomes of host cells due to its comigration with the host genome and the generation of fragments larger than the unit length of viral DNA after the digestion with SmaI (Fig. 6A and B). NotI digestion data (Fig. 6C) provided additional evidence for the insertion of viral DNA in the genomes of host cells. The combined results from the digestions with SmaI and NotI suggest that viral DNA exists as either circular or inserted forms. The circular viral DNA molecules may either exist as an episome or be encapsulated in the virion. However, viral particles were not visible under the electron microscope, suggesting that if viral particles exist during persistent viral infection, their numbers must be low.

The physical status of the viral genome was further studied by the fluorescent in situ hybridization of cells persistently infected with Hz-1 virus. Results similar to those from PFGE, i.e., the existence of extrachromosomal and inserted forms of viral genomes, were found. Hz-1 virus appears to be the first nonretroviral insect virus known to integrate its genome into host chromosomes during the infection process. The polyadnaviruses are unique insect viruses in that the genomic DNAs of the viruses are carried by the genomes of parasitoid wasps. These polyadenaviruses appear to be vertically transmitted within wasps, apparently as an integral part of their chromosomes (20). In mature females, the virus particles are released from the calyx cells into the oviduct lumen, either by budding or by cell lysis. Viruses are then injected with wasp eggs into parasitized insects (for a review, see reference 34). However, so far there is no evidence to suggest an active integration of the viral genome into host chromosomes of either the parasitic wasp or the parasitized lepidopteran larva through infection. Since the integration of Hz-1 viral DNA into an insect host chromosome can be achieved under laboratory conditions by viral infection, this model could serve as a useful tool for future engineering of insects or insect cells.

It was reported by Wood and Burand (44) that one of the persistently infected T. ni cell cultures contained approximately 500 viral genome copies per cell. The results of our dot blot hybridization experiments revealed that a host cell may harbor as much as 16% of the total DNA of the viral genome, and two of the T. ni cell lines, TNP1 and TNP3, contained very high numbers of viral copies (Table 1). Such a high viral DNA content could be burdensome to the T. ni host cells; however, these cells grew only slightly slower than the parental cells. Although the results of PFGE suggested that a portion of viral DNA may have been inserted into the host genome, it is still difficult to estimate how many copies of the viral DNA are inserted into the viral genome. Even if high copy numbers of viral DNA were inserted into the host genome, they may have been inserted as concatemers, thus reducing the insertion site and damage to host genomes.

Long-term association of Hz-1 virus and host cells has been referred to as persistent infection since the discovery of this virus (9, 22). In this study, we provided some new insights regarding the nature of persistent Hz-1 virus infection and found that persistent infection may not be the best terminology for describing such long-term Hz-1 virus-host associations. Persistent infections can be classified into three categories: latent, chronic, and slow infections. Latent infection is defined as the condition under which the virus is usually undetectable and intermittent acute symptoms may occur (44). Garcia-Blanco and Cullen (21) similarly proposed that latency is a reversible nonproductive infection of cells by a replication-competent virus. Latent infection of herpesviruses has also been defined as a type of persistent infection in which the viral genome is present but infectious virus is not produced except during intermittent episodes of reactivation (5, 40). Essentially the same definition was proposed by Ahmed (3). Similar to the case for herpesviruses, differential viral gene expression was observed in Hz-1 virus during productive and persistent viral infections (13). During persistent viral infection, the nuclear RNA PAT1 was the only virus-specific transcript that appeared to be expressed (11). In addition, infectious Hz-1 virus was not released during persistency unless a lytic reactivation took place (Fig. 1 and Table 1). These and many other features demonstrated in this study suggest that persistent infection of insect cells by Hz-1 virus is better referred to as latent viral infection.

Recently, Hughes et al. (25, 26) detected a low-level baculovirus infection in M. brassicae insects that resembles that of M. brassicae multiple nucleocapsid nuclear polyhedrosis virus. The virus was found to persist in the fat body and was detectable by PCR analysis or other sensitive indirect measures. In their work, viral protein was detectable and infectious viruses were present, suggesting that the occult virus may remain as a persistent infection. Further study of issues such as whether such occult baculovirus infection in insects is due to continuous viral production in every cell or is due to a reactivation of viruses in only some cells, and comparison of the molecular mechanism of persistent baculovirus infection with that of Hz-1 virus infection (11, 13), may generate important information for our understanding of persistent viral infection in insects.

Persistent/latent viral infection in insects is suspected to be a very common phenomenon naturally occurring in the field and in long-term laboratory colonies (9, 14, 15, 25, 28, 31, 36). Usually the virus can be easily detected during productive infection. However, between viral outbreaks, viruses may be harbored in insects as persistent/latent infections and be difficult to detect (7, 19). This study provided an opportunity for us to look further into virus-host associations in insect cells. An understanding of persistent/latent viral infections in insects may become useful in biological control programs. Furthermore, the Hz-1 virus has a broad host range and is easy to manipulate for both productive and latent infections in broad host cell lines in insects. Due to the general similarity of latent Hz-1 virus infection to latent infection by other viruses, i.e., differential viral gene expression, integration of viral DNA into the host genome, reactivation, and other characteristics, Hz-1 virus may be a useful tool for comparative studies of latent viral infections of vertebrates and invertebrates.