Hytrosaviridae: a proposal for classification and nomenclature of a new insect virus family.

Salivary gland hypertrophy viruses (SGHVs) have been identified from different dipteran species, such as the tsetse fly Glossina pallidipes (GpSGHV), the housefly Musca domestica (MdSGHV) and the narcissus bulbfly Merodon equestris (MeSGHV). These viruses share the following characteristics: (i) they produce non-occluded, enveloped, rod-shaped virions that measure 500-1,000 nm in length and 50-100 nm in diameter; (ii) they possess a large circular double-stranded DNA (dsDNA) genome ranging in size from 120 to 190 kbp and having G + C ratios ranging from 28 to 44%; (iii) they cause overt salivary gland hypertrophy (SGH) symptoms in dipteran adults and partial to complete sterility. The available information on the complete genome sequence of GpSGHV and MdSGHV indicates significant co-linearity between the two viral genomes, whereas no co-linearity was observed with baculoviruses, ascoviruses, entomopoxviruses, iridoviruses and nudiviruses, other large invertebrate DNA viruses. The DNA polymerases encoded by the SGHVs are of the type B and closely related, but they are phylogenetically distant from DNA polymerases encoded by other large dsDNA viruses. The great majority of SGHV ORFs could not be assigned by sequence comparison. Phylogenetic analysis of conserved genes clustered both SGHVs, but distantly from the nudiviruses and baculoviruses. On the basis of the available morphological, (patho)biological, genomic and phylogenetic data, we propose that the two viruses are members of a new virus family named Hytrosaviridae. This proposed family currently comprises two unassigned species, G. pallidipes salivary gland hypertrophy virus and M. domestica salivary gland hypertrophy virus, and a tentative unassigned species, M. equestris salivary gland hypertrophy virus. Here, we present the characteristics and the justification for establishing this new virus family.

Replication of the gonad-specific virus Hz-2V in Ld652Y cells mimics replication in vivo.

A newly discovered, nonoccluded insect virus, known as gonad-specific virus or Hz-2V, was found to replicate differently in two insect cell lines derived from ovarian tissues (Tn-368 cells from Trichoplusia ni and Ld652Y from Lymantria dispar). Differences between these two cell lines were observed in virus plaque forming ability, rate of viral DNA replication, time course of infectious virus production, and the mechanism of virus release from infected cells. Replication of Hz-2V in Ld652Y cells was more productive and more closely resembled in vivo virus replication.

Further characterization of the gonad-specific virus of corn earworm, Helicoverpa zea.

The gonad-specific virus (GSV) is a DNA virus infecting the reproductive tracts of adults of both sexes of the corn earworm, Helicoverpa zea, causing severe tissue deformities leading to sterility. Atypical occlusion bodies containing large concentrations of virions embedded in a granular matrix were seen in the lumen of the oviduct and the bursa copulatrix of infected females. The virus, transmitted by both sexes, was successfully propagated in vivo and in tissue culture. The GSV genome is about 225 kb in size, with no apparent similarity to the nucleopolyhedrovirus type species, AcMNPV, genomic DNA, as determined by Southern hybridization. PCR amplification of GSV genomic DNA with primers derived from the highly conserved polyhedra gene of several baculoviruses indicated no similarity. GSV at 10(-2) female equivalents (based on virus obtained from the bursa copulatrix and oviducts of one infected female) injected into a newly emerged female and mated to a normal male resulted in >95% agonadal progeny. However, at lower doses, some of the adult progeny looked normal but apparently carried a low level of the virus that could be responsible for sustenance of infection in a given colony, as well as in nature.

A field release of genetically engineered gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus (LdNPV).

The gypsy moth (Lymantria dispar L.) nuclear polyhedrosis virus was genetically engineered for nonpersistence by removal of the gene coding for polyhedrin production and stabilized using a coocclusion process. A beta-galactosidase marker gene was inserted into the genetically engineered virus (LdGEV) so that infected larvae could be tested for its presence using a colorimetric assay. In 1993, LdGEV-infected gypsy moths were released in a forested plot in Massachusetts to test for spread and persistence. A similar forested plot 2 km away served as a control. For 3 years (1993-1995), gypsy moths were established in the two plots in Massachusetts to serve as test and control populations. Each week, larvae were collected from both plots. These field-collected larvae were reared individually, checked for mortality, and then tested for the presence of beta-galactosidase. Other gypsy moth larvae were confined on LdGEV-contaminated foliage for 1 week and then treated as the field-collected larvae. The LdGEV was sought in bark, litter, and soil samples collected from each plot. To verify the presence of the LdGEV, polymerase chain reaction, slot blot DNA hybridization, and restriction enzyme analysis were also used on larval samples. Field-collected larvae infected with the engineered virus were recovered in the release plot in 1993, but not in subsequent years; no field-collected larvae from the control plot contained the engineered virus. Larvae confined on LdGEV-contaminated foliage were killed by the virus. No LdGEV was recovered from bark, litter, or soil samples from either of the plots.

Nucleotide sequence, temporal expression, and transcriptional mapping of the p34 late gene of the Hz-1 insect virus.

A late gene of the Hz-1 insect virus (Hz-1V) encoding a predicted polypeptide of 34 kilodaltons (kDa) was isolated from a cDNA library and mapped to the HindIII-T region (50.3 to 52.4 map units) of the viral genome. The p34 gene was characterized by DNA sequence, Northern blot, and primer extension analyses. The 765-bp open reading frame (ORF) is transcribed in the clockwise direction as a 1.2-kb RNA. Primer extension analysis detected two late transcription initiation sites at -16 and -17 nt relative to the start of the p34 ORF. Transcription initiation was observed between 4 and 18 hr postinfection (hr p.i.) with maximum expression at 12 hr p.i. No nucleotide sequence homology was detected between the regulatory region of the p34 gene and the baculovirus conserved late promoter motif NTAAG. This observation was substantiated by results obtained from an investigation of Hz-1V late gene expression using a transient expression assay system which suggested that Hz-1V late gene promoters do not resemble the baculovirus late promoter motif. This is the first molecular analysis of Hz-1V late gene expression and offers a basis by which to compare Hz-1V to other insect viruses.

Baculovirus replication alters hormone-regulated host development.

The baculovirus Lymantria dispar nuclear polyhedrosis virus interferes with insect larval development by altering the host's hormonal system. The level of haemolymph ecdysteroids, the insect moulting hormone, was found to be higher in virus-infected larvae than in uninfected controls. This was consistently observed in both fourth instars and day 5-infected fifth instars. The rate of hormone synthesis was examined by in vitro incubation of the prothoracic gland. Gland activity in virus-infected larvae was higher than controls and continued until the late stages of virus infection, even during the time that controls had ceased to secrete ecdysone after moulting. During virus replication, the prothoracic gland was observed to maintain morphological and ultrastructural characteristics indicative of ecdysone biosynthetic activities. Therefore, it is likely that the insects are no longer under the control of the normal hormonal system after virus infection. It is felt that the alteration of hormone titre and the rate of ecdysone synthesis is the result of the activity of ecdysteroid UDP-glucosyl transferase (EGT), a virus-encoded enzyme which is thought to inactivate ecdysteroids by sugar conjugation.

Saturable attachment sites for polyhedron-derived baculovirus on insect cells and evidence for entry via direct membrane fusion.

This research provides the first evidence for specific receptor binding of polyhedron-derived baculovirus (PDV) to host cells and to lepidopteran brush border membrane vesicles (BBMV) and demonstration of entry via a nonendocytotic pathway involving direct membrane fusion. The technique of fluorescence-activated cell sorting analysis was used to investigate the specificity of binding between the PDV phenotype of Lymantria dispar nuclear polyhedrosis virus (LdNPV) and host membranes. Fluorescein isothiocyanate-labeled PDV was found to bind in a saturable manner to the gypsy moth cell line IPLB-LdEIta and to L. dispar BBMV. The IPLB-LdEIta cell line was found to possess approximately 10(6) PDV-specific receptor sites per cell. Excess levels of unlabeled PDV were highly efficient in competing with fluorescein isothiocyanate-labeled PDV for limited receptor sites, further supporting the specificity of the interaction. Major reductions in virus binding (as high as 70%) after protease treatment of cells indicated that a protein receptor is involved. A fluorescence dequenching assay of membrane fusion with octadecyl rhodamine B (R18)-labeled PDV was used to identify PDV fusion to host cells and BBMV. Direct membrane fusion of PDV occurred at 27 degrees C to both target membranes as well as at 4 degrees C at approximately 55% of the levels achieved at 27 degrees C. Viral fusion to BBMV occurred throughout the pH range of 4 to 11, with dramatically increased fusion levels (threefold) under the alkaline conditions normal for lepidopteran larval midguts. Treatment of cells with chloroquine, a lysosomotropic agent, did not significantly affect PDV fusion to cells or infectivity in tissue culture assays.

Canine parvovirus empty capsids produced by expression in a baculovirus vector: use in analysis of viral properties and immunization of dogs.

The VP-2 genes of canine parvovirus (CPV) and a recombinant consisting of CPV and feline panleukopenia virus (FPV) sequences were cloned into baculovirus expression vectors, fused to the baculovirus polyhedrin promoter. Recombinant baculoviruses were prepared and the properties of the parvovirus proteins expressed in insect cells examined. The proteins produced were the same size as the authentic CPV VP-2 protein, and were produced late after infection; the quantity of proteins recovered from the insect cell cultures was similar to those produced in CPV infections. Parvovirus particles formed had the haemagglutination (HA), sedimentation and buoyant density properties of authentic CPV capsids. Both the CPV capsids and the CPV-FPV recombinant capsids from the baculovirus system expressed the same epitopes as those seen in the viable parvoviruses when tested with a panel of anti-parvovirus monoclonal antibodies. Lysates of recombinant baculovirus-infected cells were inoculated into dogs, giving rise to serum neutralizing and HA-inhibiting antibodies, and the immunized dogs were protected from clinical disease upon challenge with a virulent isolate of the most recent antigenic type of CPV.

The use of polymerase chain reaction and shortwave UV irradiation to detect baculovirus DNA on the surface of gypsy moth eggs.

Polymerase chain reaction (PCR) technology was employed to detect baculovirus DNA sequences from viral occlusion bodies (OB) contaminating the surface of gypsy moth eggs. The level of sensitivity of the technique was as low as 5 viral genome copies and DNA from 1 OB equivalent. Thirty minutes of shortwave UV irradiation of eggs contaminated with 8.4 x 10(4) OBs prevented amplification of viral DNA sequences from OBs on the egg surface. These methods are important for providing a better understanding of gypsy moth virus epizootiology as well as for the examination of insect eggs for the persistence of baculovirus gene sequences inside the egg or on the egg surface. In addition, these methods can be easily modified for monitoring the persistence of genetically engineered baculoviruses in insect populations as well as the fate of genes that these viruses might carry.

Field evaluation of a DNA hybridization assay for nuclear polyhedrosis virus in gypsy moth (Lepidoptera: Lymantriidae) larvae.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.

DNA hybridization assay for detection of gypsy moth nuclear polyhedrosis virus in infected gypsy moth (Lymantria dispar L.) larvae.

Radiolabeled Lymantria dispar nuclear polyhedrosis virus DNA probes were used in a DNA hybridization assay to detect the presence of viral DNA in extracts from infected larvae. Total DNA was extracted from larvae, bound to nitrocellulose filters, and assayed for the presence of viral DNA by two methods: slot-blot vacuum filtration and whole-larval squashes. To test the assays, neonate larvae were fed droplets containing a known concentration of L. dispar nuclear polyhedrosis virus and observed for up to 10 days to determine the percentage of infected larvae. The average percent mortalities were 88.0, 60.7, 26.0, and 5.3% for larvae fed droplets containing 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) polyhedral inclusion bodies (PIBs) per ml, respectively. Other larvae treated with the same virus concentrations were frozen at 2, 4, and 6 days postinoculation and examined by the hybridization techniques. The average percentage of slot blots containing viral DNA equaled 81.0, 58.0, 18.0, and 6.0% for larvae blotted 4 days after treatment with 4.0 x 10(4), 1.0 x 10(4), 2.5 x 10(3), and 6.25 x 10(2) PIBs per ml, respectively, and 89.9, 52.1, 26.6, and 6.0%, respectively at 6 days postinoculation. Thus, the hybridization results were closely correlated with mortality observed in reared larvae. Hybridization of squashes of larvae frozen 4 days after receiving the above virus treatments also produced accurate measures of the incidence of virus infection.

Interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines.

The interaction of Autographa californica nuclear polyhedrosis virus with two nonpermissive cell lines was investigated. The insect cell line, CP 169, and the Chinese hamster cell line, CHO-K1, were able to adsorb and engulf virus particles, but there was no evidence for viral replication in these cells based on virus growth titrations, electron microscopy, dot hybridization, and synthesis of viral induced proteins.

Characterization of Gypsy Moth (Lymantria dispar) Nuclear Polyhedrosis Virus.

Characterization of the proteins and nucleic acid of the gypsy moth nuclear polyhedrosis virus isolated in Ithaca, N.Y. (LdNPV-IT) is presented. A total of 29 viral structural proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the virus was isolated in the absence of alkaline protease activity. Fourteen surface envelope viral proteins were identified by lactoperoxidase iodination. Eleven proteins were associated with nucleocapsids prepared by Nonidet P-40 detergent treatment. Distinct alterations of viral proteins were documented when virions were purified in the presence of occlusion body-associated alkaline protease(s). Restriction enzyme digests of viral DNA indicated that this isolate was composed of a large number of genetic variants. On the basis of the major molar fragments resulting from EcoRI, BamHI, BglII, and HindIII digests, the molecular weight of the LdNPV genome was approximately 88 x 10.

Structural and Intracellular Proteins of the Nonoccluded Baculovirus HZ-1.

A plaque-purified isolate of the baculovirus HZ-1 was used to examine the kinetics of replication of this persistent, nonoccluded virus in TN-368 cells. Twenty-eight virus structural proteins ranging in molecular weight from 153,000 to 14,000 were identified. Fourteen of these proteins were found to be glycosylated. The sequence of appearance of the 37 virus-induced intracellular polypeptides was determined by pulse-labeling with [(35)S]methionine. N-[(3)H]acetylglucosamine, [(3)H]mannose, and the glycosylation inhibitor tunicamycin were used to detect virus structural glycoproteins. Post-transcriptional modification of two virus-induced proteins was detected.

Inhibition of Autographa californica nuclear polyhedrosis virus replication in high-density Trichoplusia ni cell cultures.

Replication of the Autographa californica nuclear polyhedrosis virus was studied in Trichoplusia ni (TN-368) tissue culture cells under cell density conditions which approximated a confluent monolayer (5.7 x 10(5) cells/cm(2) growth surface). These high-density cultures were not refractory to infection but the synthesis of both the occluded and nonoccluded forms of the virus were inhibited greater than 98% when compared to inoculation of low cell density cultures (1.4 x 10(5) cells/cm(2)). The inhibition phenomenon was not due to depletion of a medium component or a diffusable cell-associated factor. The inhibition was reversible and required cell-to-cell contact. Viral-induced protein synthesis studies using 35S-labeled methionine incorporation indicated an 18-hr delay in the synthesis of one of the four early proteins under high cell density conditions. DNA:DNA dot hybridization and 3H-labeled thymidine incorporation studies indicated that cell-to-cell contact reversibly inhibited both cellular and viral DNA synthesis.

Alteration of Autographa californica nuclear polyhedrosis virus DNA upon serial passage in cell culture.

Plaque-purified Autographa californica nuclear polyhedrosis virus, AcMNPV E2 isolate, was propagated for 30 generations by serial passage in TN-368 cells. After passage, the DNAs from 7 of 20 replaque-purified isolates were found to have additional EcoRI restriction enzyme fragments. These 7 isolates were of three types. The additional EcoRI fragments of each isolate hybridized to existing viral DNA. Restriction enzyme mapping with SacI and SmaI show that isolates of each type contain an insertion of repeated viral DNA in approximately the same region of the genome. The origin and location of this DNA is discussed.

Replication of Heliothis zea baculovirus in an insect cell line.

The baculovirus, Heliothis zea nuclear polyhedrosis virus (HzSNPV), was grown in the H. zea cell line, IPLB-1075. Light microscopy observations showed that 95% of inoculated cells had CPE by 6 days postinoculation (p.i.). Growth curves of intracellular and extracellular virus showed that maximum titers (TCID50 units) were reached 3-4 days p.i. Studies on HzSNPV morphogenesis revealed that ultrastructural events were typical of baculovirus replication in vitro. EcoRI restriction enzyme analyses of viral DNA produced in larvae and in cell culture were identical and indicated that this virus was HzSNPV.

Physical Maps of Autographa californica and Rachiplusia ou Nuclear Polyhedrosis Virus Recombinants.

TN-368 cells were infected simultaneously with the closely related Autographa california (AcMNPV) and Rachiplusia ou (RoMNPV) nuclear polyhedrosis viruses. Progeny viral isolates were plaque purified, and their DNAs were analyzed with restriction endonucleases. Of 100 randomly cloned plaques, 7 were AcMNPV and RoMNPV recombinants, 5 were RoMNPV, and 88 were AcMNPV. The recombinants contained DNA sequences derived from both parental genomes. By comparing the restriction cleavage patterns of parental and recombinant DNAs, the crossover sites were mapped. A single double crossover was detected in each of the seven recombinant genomes. In addition, six of the seven recombinants revealed a crossover site mapping between 78 and 89% of the genome. The structural polypeptides of the seven recombinants and two parental viruses were analyzed by polyacrylamide gel electrophoresis, and their polyhedrins were identified by tryptic peptide mapping. An analysis of the segregation of three enveloped nucleocapsid proteins and of the polyhedrins among the recombinants located the DNA sequences coding for AcMNPV structural polypeptides with molecular weights of 37,000 (a capsid polypeptide), 56,000, and 90,000 and the RoMNPV structural polypeptides with molecular weights of 36,000 (a capsid polypeptide), 56,000, and 91,000. The AcMNPV and RoMNPV polypeptides of molecular weights 37,000 and 36,000, respectively, mapped within 78 to 89% or 1 to 29%, the polypeptides of molecular weights 55,000 and 56,000 mapped within 78 to 29%, and the polypeptides of molecular weights 90,000 and 91,000 mapped within 19 to 56% of the genome. The region of the parental DNAs that codes for polyhedrin was located within 70 to 89% of the genome.

Transfection with baculovirus DNA.

Purified DNA from the nuclear polyhedrosis viruses of Autographa californica (AcM NPV) and Rachiplusia ou (RoMNPV) were found to be infectious in TN-368 cells employing the calcium phosphate precipitation technique (F. L. Graham and A. J. van der Eb, Virology, 52 ,456-467, 1973). Transfection with AcM NPV produced 3600 PFU/microg DNA compared to 2900 PFU/mug DNA with RoM NPV. Of eight baculovirus DNAs tested, only AcM NPV DNA and RoM NPV DNA could transfect TN-368 cells. The in vitro host range of AcM NPV DNA was determined to be the same as AcM NPV extracellular virus. AcM NPV Form I DNA was fourfold more infectious in TN-368 cells than Form II DNA.