Production of polyhedral inclusion bodies from Helicoverpa armigera larvae infected with wild-type and recombinant HaSNPV

We report on the yield of Polyhedral Inclusion Bodies (PIBs) from first to fifth instar Helicoverpa armigera larvae inoculated with wild-type Helicoverpa armigera nucleopolyhedrovirus (HaSNPV-WT) or with one of two HaSNPV recombinants, one in which the ecdysteroid UDP-glucosyltransferase (egt) gene was deleted (HaSNPV-EGTD) and a second in which the egt gene was replaced by a scorpion toxin (AaIT) gene (HaSNPV-AaIT). A significant linear relationship between the logarithm of cadaver weight and the logarithm of the number of PIBs per cadaver was observed for all three virus types. The increase of the number of PIBs with larval weight was significantly greater for HaSNPV-WT than for the recombinant viruses. For each of the three HaSNPVs, PIB yield per cadaver was significantly affected by larval instar at death and by time to death, with later instars and longer surviving larvae producing a greater number of PIBs. As both recombinants caused host larvae to die at earlier instars than HaSNPV-WT, their virus yields were significantly reduced. Virus yield per larva, inoculated with HaSNPV-AaIT in the first, second, third, fourth or fifth larval stage was 23, 32, 41, 44 and 47% of the yield of HaSNPV-WT, respectively. For HaSNPV-EGTD, virus yield per larva inoculated in first through fifth instar, respectively, was 41, 55, 63, 54 and 82% of the yield of HaSNPV-WT. These results provide a basis for optimizing the production regime of recombinant HaSNPVs in larvae and for modeling the behavior of these viruses in agro-ecosystems     

Field inactivation of wild-type and genetically modified Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus in cotton

Cotton bollworm (Helicoverpa armigera) is a serious pest on cotton in China. A specific baculovirus, H. armigera nucleopolyhedroviruses (HaSNPV) is used as a commercial biopesticide to control this pest. To improve the pesticidal properties, HaSNPV has been genetically engineered by both deleting the ecdysteroid UDP-glucosyltransferase (egt) gene from its genome (recombinant HaSNPV-EGTD) and incorporating an insect-selective toxin gene from the scorpion Androctonus australis (AaIT) (recombinant HaSNPV-AaIT). In the field, there was no significant difference among the inactivation rates of the two recombinant HaSNPVs and their parent wild-type, HaSNPV-WT. The inactivation rate of these viruses was significantly different in different years. The average half-life of HaSNPV was 0.57, 0.90 and 0.39 days in 2000, 2001 and 2002, respectively. Inactivation rates correlated well with solar radiation over these years.     

Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been developed as a commercial biopesticide to control the cotton bollworm, H. armigera, in China. The major limitation to a broader application of this virus has been the relative long time to incapacitate the target insect. Two HaSNPV recombinants with improved insecticidal properties were released in bollworm-infested cotton. One recombinant (HaCXW1) lacked the ecdysteroid UDP-glucosyltransferase (egt) gene and in another recombinant (HaCXW2), an insect-selective scorpion toxin (AaIT) gene replaced the egt gene. In a cotton field situation H. armigera larvae treated with either HaCXW1 or HaCXW2 were killed faster than larvae in HaSNPV-wt treated plots. Second instar H. armigera larvae, which were collected from HaCXW1 and HaCXW2 treated plots and further reared on artificial diet, showed reduced ST(50) values of 15.3 and 26.3%, respectively, as compared to larvae collected from HaSNPV-wt treated plots. The reduction in consumed leaf area of field collected larvae infected with HaCXW1 and HaCXW2 was approximated 50 and 63%, respectively, as compared to HaSNPV-wt infected larvae at 108 h after treatment. These results suggest that in a cotton field situation the recombinants will be more effective control agents of the cotton bollworm than wild-type HaSNPV.     

Horizontal and vertical transmission of viruses in the honey bee, Apis mellifera

The most crucial stage in the dynamics of virus infections is the mode of virus transmission. In general, transmission of viruses can occur through two pathways: horizontal and vertical transmission. In horizontal transmission, viruses are transmitted among individuals of the same generation, while vertical transmission occurs from mothers to their offspring. Because of its highly organized social structure and crowded population density, the honey bee colony represents a risky environment for the spread of disease infection. Like other plant and animal viruses, bee viruses use different survival strategies, including utilization of both horizontal and vertical routes, to transmit and maintain levels in a host population. In this review, we explore the current knowledge about the honey bee viruses and transmission routes of bee viruses. In addition, different transmission strategies on the persistence and dynamics of host-pathogen interactions are also discussed.     

Infectivity, speed of kill, and productivity of a baculovirus expressing the itch mite toxin txp-1 in second and fourth instar larvae of Trichoplusia ni

A cDNA clone of the gene coding for the paralytic neurotoxin (tox34) from the female straw itch mite, Pyemotes tritici, was created by RT-PCR and inserted into the genome of the Autographa californica nucleopolyhedrovirus (AcMNPV) under the control of the AcMNPV p10 promoter. This recombinant virus, AcTOX34.4, caused a rigid paralysis in infected larvae. The infectivity of AcTOX34.4 was compared to the wild-type parent strain, AcMNPV-C6, in second and fourth instar larvae of the cabbage looper, Trichoplusia ni. There were no significant differences in LD(50) values between the recombinant virus and its wild-type parent strain but, as expected, the LD(50) was lower for second instar larvae. The mean time to death and yield of occlusion bodies were measured in second and fourth instar T. ni larvae at a high (100% mortality) and low (<50% mortality) doses of the virus. The mean time to death of recombinant infected larvae was reduced by 50-60% compared to larvae infected with the wild-type strain, depending on virus dose and instar, with these larvae becoming paralysed after approximately 60 h and dying 10-20 h later. This is among the fastest speeds of kill recorded for recombinant baculoviruses. Fourth instar larvae were found to succumb to the recombinant virus more quickly than the second instar larvae. The increase in the speed of kill of the recombinant virus was accompanied by a large reduction of approximately 95% in the yield of progeny virus. The yield of virus showed a highly significant relationship with time to death, but this relationship was complex and varied between the different viruses, concentrations, and instars. The yield per unit weight of the larvae was found to be constant at a low virus dose and increased over time at a high virus dose, irrespective of instar and virus. It is predicted that these changes in the performance of the recombinant virus would act toward reducing its fitness, leading to it being outcompeted by the wild type in field situations.     

Transmission of NPV in uniform- and mixed-age populations ofHeliothis zea [Lep: Noctuidae] on caged soybean

Transmission of a nuclear polyhedrosis virus (NPV) was examined in uniformaged and uniform- vs mixed-age populations ofHeliothis zea (Boddie) on caged soybean. Larval collections revealed viral disease outbreaks occurred in all treatments following release of infected larvae (Primary infected larvae). Transmission of NPV in uniform-aged populations was related to the density of primary infected larvae released in the population but not to the size at death of primary infected larvae (P<0.05). In mixed-age populations horizontal transmission in the oldest larvae in the population was equal to that in uniform-aged populations, providing that primary infected larvae in the mixed-aged population were all the age of the oldest noninfected cohorts. As the mixed-age population aged, transmission increased and was generally higher than that in the uniform-aged populations. Transmission was also higher when primary infected larvae were medium sized at death than when small or large at death. The concentration of virus deposited on foliage and in soil after all larvae had died on plants was related to density of primary infected larvae released. In soil, but not on foliage, the virus concentration was related to the size at death of primary infected larvae released. This material is based upon work supported in part by the U.S. Department of Agriculture under Agreement No. CRSR-2-1000.     

Parasitoid-mediated transmission of an iridescent virus

We examined the interaction between an invertebrate iridescent virus (IIV) isolated from Spodoptera frugiperda (J.E. Smith) and the solitary ichneumonid endoparasitoid Eiphosoma vitticolle Cresson. In choice tests, parasitoids examined and stung significantly more virus infected than healthy larvae, apparently due to a lack of defense reaction in virus infected hosts. Parasitoid-mediated virus transmission was observed in 100% of the female parasitoids that stung a virus infected host in the laboratory. Each female parasitoid transmitted the virus to an average (+/-SE) of 3.7+/-0.3 larvae immediately after stinging an infected larva. Caged field experiments supported this result; virus transmission to healthy larvae only occurred in cages containing infected hosts (as inoculum) and parasitoids (as vectors). The virus was highly detrimental to parasitoid development because of premature host death and lethal infection of the developing endoparasitoid. Female parasitoids that emerged from virus infected hosts did not transmit the virus to healthy hosts. We suggest that the polyphagous habits of many noctuid parasitoids combined with the catholic host range of most IIVs may represent a mechanism for the transmission of IIVs between different host species in the field.     

Quantifying horizontal transmission of Nosema lymantriae, a microsporidian pathogen of the gypsy moth, Lymantria dispar (Lep., Lymantriidae) in field cage studies

Nosema lymantriae is a microsporidian pathogen of the gypsy moth, Lymantria dispar that has been documented to be at least partially responsible for the collapse of L. dispar outbreak populations in Europe. To quantify horizontal transmission of this pathogen under field conditions we performed caged-tree experiments that varied (1) the density of the pathogen through the introduction of laboratory-infected larvae, and (2) the total time that susceptible (test) larvae were exposed to these infected larvae. The time frame of the experiments extended from the early phase of colonization of the target tissues by the microsporidium to the onset of pathogen-induced mortality or pupation of test larvae. Upon termination of each experiment, the prevalence of infection in test larvae was evaluated. In the experiments performed over a range of pathogen densities, infection of test larvae increased with increasing density of inoculated larvae, from 14.2 ± 3.5% at density of 10 inoculated per 100 larvae to 36.7 ± 5.7% at 30 inoculated per 100 larvae. At higher densities, percent infection in test larvae appeared to level off (35.7 ± 5.5% at 50 inoculated per 100 larvae). When larval exposure to the pathogen was varied, transmission of N. lymantriae did not occur within the first 15 d post-inoculation (dpi) (11 d post-exposure of test larvae to inoculated larvae). We found the first infected test larvae in samples taken 20 dpi (16 d post-exposure). Transmission increased over time; in the cages sampled 25 dpi (21 d post-exposure), Nosema prevalence in test larvae ranged from 20.6% to 39.2%.     

Horizontal transmission of Beauveria bassiana (Bals.) Vuill

1 Factors influencing horizontal transmission of the entomopathogen Beauveria bassiana in the Colorado potato beetle (CPB) were examined through a series of laboratory studies. 2 Cadaver density, cadaver life stage, ambient temperature and conidial density were the factors manipulated. 3 Mortality and sporulation of burrowing CPB prepupae both increased significantly with increased sporulating second‐instar cadaver density on the soil surface. 4 Mortality rates were significantly higher when prepupae were released into laboratory arenas containing third‐instar cadavers compared to second‐instar cadavers. 5 Mortality and sporulation decreased significantly as temperature increased from 15 °C to 30 °C, however, no temperature‐dependent behavioural response by prepupae could be identified as a potential cause. 6 An 86.1% decrease in conidial density per cadaver had no significant effect on mortality or sporulation of prepupae, indicating that this level of environmental degradation of cadavers may not significantly reduce the probability of horizontal transmission.     

Digestive proteolytic and amylolytic activities and feeding responses of Helicoverpa armigera (Lepidoptera: Noctuidae) on different host plants

Digestive proteolytic and amylolytic activities and feeding responses of fifth instar larvae of Helicoverpa armigera (Hübner) on different host plants including chickpea (cultivars Arman, Hashem, Azad, and Binivich), common bean (cultivar Khomein), white kidney bean (cultivar Dehghan), red kidney bean (cultivar Goli), cowpea (cultivar Mashhad), tomato (cultivar Meshkin), and potato (cultivars Agria and Satina) were studied under laboratory conditions (25 +/- 1 degrees C, 65 +/- 5% RH and a photoperiod of 16:8 [L:D] h). Our results showed that the highest protease activity in optimal pH was on cultivar Dehghan (8.717 U/mg) and lowest one was on Meshkin (3.338 U/mg). In addition, the highest amylase activity in optimal pH was on cultivar Dehghan (0.340 mU/mg) and lowest was on Meshkin (0.088 mU/mg). The larval weight of fifth instar H. armigera showed significant difference, being heaviest on Binivich (125.290 +/- 5.050 mg) and lightest on Meshkin (22.773 +/- 0.575 mg). Furthermore, the highest and lowest values of food consumed were on Goli (362.800 +/- 27.500 mg) and Satina (51.280 +/- 4.500 mg), respectively. In addition, the lowest values of prepupal and pupal weight were on Meshkin (32.413 +/- 0.980 and 41.820 +/- 1.270 mg, respectively). The results indicated that tomato (Meshkin) was unsuitable host for feeding fifth instar larvae of H. armigera.     

Natural vertical transmission of dengue viruses by Aedes aegypti in Bolivia

The natural transmission of dengue virus from an infected female mosquito to its progeny, namely the vertical transmission, was researched in wild caught Aedes aegypti during an important outbreak in the town of Santa Cruz de la Sierra, Bolivia. Mosquitoes were collected at the preimaginal stages (eggs, larvae and pupae) then reared up to adult stage for viral detection using molecular methods. Dengue virus serotypes 1 and 3 were found to be co-circulating with significant higher prevalence in male than in female mosquitoes. Of the 97 pools of Ae. aegypti (n = 635 male and 748 female specimens) screened, 14 pools, collected in February-May in 2007, were found positive for dengue virus infection: five DEN-1 and nine DEN-3. The average true infection rate (TIR) and minimum infection rate (MIR) were respectively 1.08% and 1.01%. These observations suggest that vertical transmission of dengue virus may be detected in vectors at the peak of an outbreak as well as several months before an epidemic occurs in human population.     

Virulence evolution in a virus obeys a trade-off

The evolution of virulence was studied in a virus subjected to alternating episodes of vertical and horizontal transmission. Bacteriophage f1 was used as the parasite because it establishes a debilitating but non-fatal infection that can be transmitted vertically (from a host to its progeny) as well as horizontally (infection of new hosts). Horizontal transmission was required of all phage at specific intervals, but was prevented otherwise. Each episode of horizontal transmission was followed by an interval of obligate vertical transmission, followed by an interval of obligate horizontal transmission etc. The duration of vertical transmission was eight times longer per episode in one treatment than in the other, thus varying the relative intensity of selection against virulence while maintaining selection for some level of virus production. Viral lines with the higher enforced rate of infectious transmission evolved higher virulence and higher rates of virus production. These results support the trade-off model for the evolution of virulence.     

Effect of starvation upon baculovirus replication in larval Bombyxmori and Heliothisvirescens

The progression of baculovirus (BmNPV, BmCysPD, AcMNPV or AcAaIT) infection in larval Bombyx mori and Heliothis virescens (1st, 3rd or 5th instar) was investigated following various starvation regimes. When the larvae were starved for 12 or 24 h immediately following inoculation, the median lethal time to death (LT₅₀) was delayed by 9.5-19.2 h in comparison to non-starved controls. This corresponded to a delay of 10-23% depending upon the larval stage and virus that was used for inoculation. When a 24 h-long starvation period was initiated at 1 or 2 days post inoculation (p.i.), a statistically significant difference in LT₅₀ was not found indicating that the early stages of infection are more sensitive to the effects of starvation. Viral titers in the hemolymph of 5th instar B. mori that were starved for 24 h immediately following inoculation were 10-fold lower (p < 0.01) than that found in non-starved control larvae. Histochemical analyses indicated that virus transmission was reduced in 5th instar B. mori that were starved for 24 h immediately following inoculation in comparison to non-starved control larvae. In general, the mass of larvae that were starved immediately after inoculation was 30% lower than that of non-starved control insects. Our findings indicate that starvation of the larval host at the time of baculovirus exposure has a negative effect on the rate baculovirus transmission and pathogenesis.     

In vivo enhancement of nucleopolyhedrovirus of oriental Armyworm, Mythimna separata using spindles from Helicoverpa armigera entomopoxvirus

When the third instar larvae of M. separata were exposed to eight varying concentrations of polyhedral occlusion bodies (POB's) of nucleopolyhedrovirus of M. separata (MsNPV) ranging from 2.6 x 10(-1) to 2.6 x 10(8) POB's/ml, the percent mortality and incubation period ranged from 16-100% and 14 to 9 days respectively. On the other hand when the same third instar larvae of M. separata were exposed to only five varying concentration of POB's of MsNPV ranging from 2.6 x 10(2) to 2.6 x 10(6), POB's/ml along with a constant dose of entomopox viral spindles from Helicoverpa armigera, the per cent mortality ranged from 63 to 100% with reduction in incubation period from 7 to 4 days respectively. The enhancement index (log10) of the virus was 2.76 or reduction of more than 500 times in LC50. The ability and the mechanism of the spindles from H. armigera entomopoxvirus to enhance the infectivity of MsNPV has been discussed.     

Low frequency of horizontal and vertical transmission of two begomoviruses through whiteflies exhibits little relevance to the vector infectivity

Transmissions of plant viruses between individuals of their vector insects through mating are rare events. Recently, three begomoviruses were found to be transmitted between males and females of the whitefly Bemisia tabaci through mating, and two viruses were shown to be transmitted transovarially to progeny. However, results between reports were not consistent. Here we examined the horizontal and vertical transmission of Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl China virus (TYLCCNV) by the B and Q biotypes of B. tabaci, using virus isolates and whitefly colonies established recently in China. Both TYLCV DNA and TYLCCNV DNA were shown to be transmitted horizontally and vertically by each of the two biotypes of the whitefly, but frequency of transmission was usually low. In transovarial transmission, virus DNA was detected in eggs and nymphs but not in the adults of the first generation progeny, except in the combination of TYLCV and Q biotype whitefly where 2–3% of the offspring adults contained the virus DNA. We also showed that the first generation adults, which developed from eggs of viruliferous whiteflies, were not infective to plants. These results demonstrated that for the viruses and whiteflies tested here low frequency of horizontal and vertical transmission can be expected but these two modes of transmission are unlikely to have much epidemiological relevance in the field.     

平菇病毒dsRNA传播途径的探究

通过对平菇孢子dsRNA检测、单双杂交和单单杂交,从垂直传播和水平传播2个方面来研究平菇病毒dsRNA的传播途径,结果表明：病毒dsRNA通过孢子垂直传播的几率很小,通过细胞融合的水平传播几率相对比较高,在实际生产中平菇病毒dsRNA的主要传播途径还有待进一步的研究。     

颗粒体病毒在小菜蛾种群中的垂直传播方式的初步观察

在室内观察了小菜蛾颗粒体病毒PlutellaxylostellaGranulosisVirus(RxGV)在小菜蛾种群水平上可能的垂直传播途径。结果表明 :(1 )上代小菜蛾Plutellaxylostella(L .)幼虫期饲毒不能将小菜蛾颗粒体病垂直传递到下代 ;(2 )取食病毒的雌蛾产的卵所孵化的幼虫中 ,没病毒感染出现 ,说明病毒不能经小菜蛾成虫体内传播到下一代。 (3 )用小菜蛾颗粒体病毒处理卵卡时 ,其初孵幼虫有少量被病毒感染死亡 ,浓度高时感染率也较高 ,但感染率均在 1 0 %以下 ,说明小菜蛾颗粒体病毒能经卵表垂直传递给下代 ,但传递效率不高。但当用病毒处理叶片上的卵时 ,初孵幼虫死亡率高达 77 73 % ,而且孵化出的幼虫大多数染病且死于 3龄以前 ,因此田间病毒的施用时间可提早到卵高峰期。     

Vertical transmission of TnSNPV,TnCPV, AcMNPV,and possibly recombinant NPV in Trichoplusia ni

Four viruses were tested for vertical transmission in Trichoplusia ni: T. ni nucleopolyhedrovirus (TnSNPV), T. ni cypovirus (TnCPV), Autographa californica nucleopolyhedrovirus (AcMNPV), and AcMNPV engineered to express a scorpion toxin (AcMNPV.AaIT). Fifth instars were exposed to each virus, the survivors were reared and mated, and second-generation (F(1)) insects were examined for infection. TnSNPV was transmitted to offspring at a prevalence rate of 15.4%, TnCPV at 10.2%, and AcMNPV at 10.1%. Only one of 2484 F(1) insects was infected with AcMNPV.AaIT; this experiment was repeated, and none of 4774 insects was infected. Thus, vertical transmission is unlikely to contribute to AcMNPV.AaIT contacting non-target organisms after its field release. There was evidence that TnCPV and possibly TnSNPV were activated to overt infections by ingestion of a different virus. TnCPV, but not the NPVs, routinely infected 0.3-1.7% of non-treated insects, probably indicating that it is vertically transmitted at enzootic levels.     

Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins

Intra-specific variation in susceptibility of Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) in Australia to the Cry1Ac and Cry2Ab delta-endotoxins from Bacillus thuringiensis (Berliner) (Bt) was determined to establish a baseline for monitoring changes that might occur with the use of Bt cotton. Strains of H. armigera and H. punctigera were established from populations collected primarily from commercial farms throughout the Australian cotton belts. Strains were evaluated for susceptibility using two bioassay methods (surface treatment and diet incorporation) by measuring the dose response for mortality (LC50) and growth inhibition (IC50). The variation in LC50 among H. armigera (n=17 strains) and H. punctigera (n=12 strains) in response to Cry1Ac was 4.6- and 3.2-fold, respectively. The variation in LC50 among H. armigera (n=19 strains) and H. punctigera (n=12 strains) to Cry2Ab was 6.6- and 3.5-fold, respectively. The range of Cry1Ac induced growth inhibition from the 3rd to 4th instar in H. armigera (n=15 strains) was 3.6-fold and in H. punctigera (n=13 strains) was 2.6-fold, while the range of Cry2Ab induced growth inhibition from neonate to 3rd instar in H. armigera (n=13 strains) was 4.3-fold and in H. punctigera (n=12 strains) was 6.1-fold. Variation in susceptibility was also evaluated for two age classes (neonates and 3rd instars) in laboratory strains of H. armigera and H. punctigera. Neonates of H. punctigera had the same or higher sensitivity to Bt than 3rd instars. Neonates of H. armigera were more sensitive to Cry2Ab than 3rd instars, while being less sensitive to Cry1Ac than 3rd instars. Differences in the two methods of bioassay used affected relative sensitivity of species to Bt toxins, highlighting the need to standardize bioassay protocols.     

Father-to-Mother-to-Infant Transmission of HIV-1: Clonally Transmitted Isolate of Infant Mutates More Rapidly than That of the Mother and Rapidly Loses Reactivity with Neutralizing Antibody

The sequences of the V3 loop and surrounding regions of human immunodeficiency virus type-1 from a father-to-mother-to-infant trimmer were studied and the horizontal and vertical transmissions compared. The father's virus was variable for reactivity with neutralizing antibody and sequences of the V3 loop central core sequence. In contrast, the mother's viral sequences were much less diverse and reacted with a virus neutralizing antibody. The infant's viral sequences were also less diverse than those of the father, and N-glycosylation sites were conserved. By phylogenetic analysis, the major clone, of which V3-peptide reacted with the neutralizing antibody, was found to be transmitted from the mother to her infant; however, the mutated minor clones did not bind to the antibody. These findings suggest that both horizontal and vertical virus transmission were selective, and that the clonally transmitted virus in infants mutates more rapidly than viruses in the mother, to whom the virus was horizontally transmitted.