Persistence of naturally occurring and genetically modified Choristoneura fumiferana nucleopolyhedroviruses in outdoor aquatic microcosms.

BACKGROUND: To assess the persistence of genetically modified and naturally occurring baculoviruses in an aquatic environment, replicate (three) outdoor, aquatic microcosms were spiked with spruce budworm viruses [Ireland strain of Choristoneura fumiferana multiple nucleopolyhedrovirus (CfMNPV) and the recombinant CfMNPVegt(-)/lacZ(+)] at a rate of 1.86 x 10(10) occlusion bodies (OBs) m(-2) of surface area. The presence of virus in water samples collected at various times after inoculation was determined by PCR amplification of baculoviral DNA extracted from OBs. RESULTS: Although UV radiation rapidly degrades baculoviruses under natural conditions, both viruses persisted above the level of detection (>100 OBs 450 microL(-1) of natural pond water) for at least 1 year post-inoculation, with little difference between the viruses in their patterns of persistence. CONCLUSION: The present microcosm study suggests that occlusion bodies of baculoviruses can persist in the flocculent layer of natural ponds. On disturbance, OBs could re-enter the main water column and thus be available for transport to new locations. Implications for environmental risk assessment are discussed.

Comparative activity of Choristoneura fumiferana nucleopolyhedrovirus propagated in different hosts.

The biological activity of the Ireland strain of Choristoneura fumiferana (Clem) nucleopolyhedrovirus (CfMNPV) propagated in different hosts was determined to provide the basis upon which genetically modified CfMNPV, or other naturally occurring isolates, should be compared. Occlusion bodies (OB) derived from CF-203 cells were significantly larger and more pathogenic than those propagated in vivo when tested against the fifth larval instar of C fumiferana (Clem) and C occidentalis Freeman. The dose-responses (LD50 and LD95, expressed as occlusion bodies per larva) of C fumiferana larvae to in vitro-propagated OBs were 274 and 5785, respectively. The values of LD50 and LD95 to C occidentalis larvae were 19 and 118, respectively. There were no significant differences in pathogenicity or size when OBs propagated in C fumiferana larvae were tested against either insect species, nor were there significant differences for OBs propagated in C occidentalis larvae. The LD50 and LD95 of in vivo-produced OBs to C fumiferana were 925 and 61988, respectively. The LD50 and LD95 to C occidentalis were 50 and 453, respectively. OBs propagated in vitro had a mean volume of 13.13 microm3, whereas those propagated in vivo ranged from 0.84 to 1.41 microm3. The median survival time-responses (ST50) of fifth-instar C fumiferana or C occidentalis larvae to OBs propagated in vivo were not significantly different from those propagated in vitro at the dosage levels tested. Values of ST50 of C fumiferana larvae to in vitro- and in vivo-produced OBs at dosages causing less than 50% mortality rangedfrom 9.6 to 9.8 days post-inoculation (dpi), whereas a LD95 dose resulted in ST50 values ranging from 7.3 to 7.7 days. ST50 values of C occidentalis larvae at dosages causing less than 50% mortality ranged from 9.8 to 10.2 dpi, whereas a LD95 dose resulted in ST50 values ranging from 9.5 to 9.8 dpi. The median feeding cessation time-response (FT50) of fifth-instar C fumiferana larvae to OBs propagated in vitro (5.7 days) was not significantly different from the FT50 of those propagated in vivo in either insect species (5.3 and 5.7 days) at the dosage level tested (LD95). No significant differences in FT50 values were observed between OBs propagated in either larval host. The FT50 of C occidentalis larvae to OBs propagated in vitro (7.7 days) was not significantly different from that to those propagated in vivo in C occidentalis larvae (7.6days), but somewhat different (7.2 days) from that to those propagated in C fumiferana larvae. Results indicate that CfMNPV can be propagated in vivo in either C fumiferana or C occidentalis larvae (or sequentially through both) without alteration in infectivity, although the use of the CF-203 cell line yields the most biologically active OBs.

Pathogenicity of Choristoneura fumiferana nucleopolyhedrovirus propagated in vitro at different incubation temperatures.

To optimize the in vitro production of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) as a potential microbial pest control agent, the pathogenicity of occlusion bodies (OBs) produced in two cell lines at three incubation temperatures was determined by bioassay. A plaque-purified isolate of CfMNPV was amplified in permissive C. fumiferana cell lines, FPMI-CF-203 and FPMI-CF-2C1, and incubated at 22, 24, and 28 degrees C. Occlusion bodies propagated in FPMI-CF-203 cells at 28 degrees C were significantly larger (17.5 microm(3)) and more pathogenic (LD(50) = 27; LD(95) = 185, where LD(50) and LD(95) are doses required to kill 50 and 95% of the test larvae, respectively) than those produced in either of the cell lines at any of the incubation temperatures tested. Increased temperatures yielded larger OBs from both cell lines. The pathogenicity of OBs propagated in the FPMI-CF-203 cell line increased with incubation temperature, whereas that of OBs produced in FPMI-CF-2C1 cells decreased. Comparison of the pathogenicity of OBs, whether naturally occurring or genetically modified, should be standardized by cell line and incubation temperature used for propagation. Production efficiency decreased with increasing incubation temperature for each cell line. Lower incubation temperatures used for propagation, and standardization of the titer of viral inoculum, should be further investigated to determine the economic feasibility of the in vitro production of CfMNPV as a microbial pest control agent.

A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats.

A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (OB) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. OBs, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5 g of lyophilized L, F-H and mineral soil horizons, and 1.0 ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0 ml of pond water and 1.0 g of bottom sediment, respectively.