Pathogenic Assessment of SfMNPV-Based Biopesticide on Spodoptera frugiperda (Lepidoptera: Noctuidae) Developing on Transgenic Soybean Expressing Cry1Ac Insecticidal Protein.

Pathogenic assessment of a baculovirus-based biopesticide containing Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV: Baculoviridae: Alphabaculovirus) infecting fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) is reported. In the bioassays, neonates were infected with different doses of SfMNPV applied on Cry1Ac Bt soybean and non-Bt soybean. Our findings indicated that S. frugiperda neonates did not survive at 10 d post infection or develop into adults on Bt and non-Bt soybean sprayed with the field recommended dose of SfMNPV. In contrast, a proportion of the infected neonates developed into adults when infected with lower doses of SfMNPV (50%, 25%, and 10% of field dose) in both Bt and non-Bt soybean. However, S. frugiperda neonates surviving infection at the lowest virus doses on both soybean varieties showed longer neonate-to-pupa and neonate-to-adult periods, lower larval and pupal weights, reduced fecundity, and increased population suppression. Nevertheless, more pronounced pathogenicity of SfMNPV infecting neonates of S. frugiperda were verified on larvae that developed on Bt soybean. These findings revealed that, beyond mortality, the biopesticide containing SfMNPV also causes significant sublethal pathogenic effects on neonates of S. frugiperda developing on Bt and non-Bt soybean and suggested an additive effect among SfMNPV and Cry1Ac insecticidal protein expressed in Bt soybean.

Rapid and cost-effective process based on insect larvae for scale-up production of SARS-COV-2 spike protein for serological COVID-19 testing.

Serology testing for COVID-19 is important in evaluating active immune response against SARS-CoV-2, studying the antibody kinetics, and monitoring reinfections with genetic variants and new virus strains, in particular, the duration of antibodies in virus-exposed individuals and vaccine-mediated immunity. In this study, recombinant S protein of SARS-CoV-2 was expressed in Rachiplusia nu, an important agronomic plague. One gram of insect larvae produces an amount of S protein sufficient for 150 determinations in the ELISA method herein developed. We established a rapid production process for SARS-CoV-2 S protein that showed immunoreactivity for anti-SARS-CoV-2 antibodies and was used as a single antigen for developing the ELISA method with high sensitivity (96.2%) and specificity (98.8%). Our findings provide an efficient and cost-effective platform for large-scale S protein production, and the scale-up is linear, thus avoiding the use of complex equipment like bioreactors.

A detailed model and Monte Carlo simulation for predicting DIP genome length distribution in baculovirus infection of insect cells.

Baculoviruses have enormous potential for use as biopesticides to control insect pest populations without the adverse environmental effects posed by the widespread use of chemical pesticides. However, continuous baculovirus production is susceptible to DNA mutation and the subsequent production of defective interfering particles (DIPs). The amount of DIPs produced and their genome length distribution are of great interest not only for baculoviruses but for many other DNA and RNA viruses. In this study, we elucidate this aspect of virus replication using baculovirus as an example system and both experimental and modeling studies. The existing mathematical models for the virus replication process consider DIPs as a lumped quantity and do not consider the genome length distribution of the DIPs. In this study, a detailed population balance model for the cell-virus culture is presented, which predicts the genome length distribution of the DIP population along with their relative proportion. The model is simulated using the kinetic Monte Carlo algorithm, and the results agree well with the experimental results. Using this model, a practical strategy to maintain the DIP fraction to near to its maximum and minimum limits has been demonstrated.

Host permissiveness to baculovirus influences time-dependent immune responses and fitness costs.

Insects possess specific immune responses to protect themselves from different types of pathogens. Activation of immune cascades can inflict significant developmental costs on the surviving host. To characterize infection kinetics in a surviving host that experiences baculovirus inoculation, it is crucial to determine the timing of immune responses. Here, we investigated time-dependent immune responses and developmental costs elicited by inoculations from each of two wild-type baculoviruses, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Helicoverpa zea single nucleopolyhedrovirus (HzSNPV), in their common host H. zea. As H. zea is a semi-permissive host of AcMNPV and fully permissive to HzSNPV, we hypothesized there are differential immune responses and fitness costs associated with resisting infection by each virus species. Newly molted 4th-instar larvae that were inoculated with a low dose (LD15 ) of either virus showed significantly higher hemolymph FAD-glucose dehydrogenase (GLD) activities compared to the corresponding control larvae. Hemolymph phenoloxidase (PO) activity, protein concentration and total hemocyte numbers were not increased, but instead were lower than in control larvae at some time points post-inoculation. Larvae that survived either virus inoculation exhibited reduced pupal weight; survivors inoculated with AcMNPV grew slower than the control larvae, while survivors of HzSNPV pupated earlier than control larvae. Our results highlight the complexity of immune responses and fitness costs associated with combating different baculoviruses.

Statistical modeling of cell-to-cell variability in viral infection during passaging in suspension cell culture: Application in Monte-Carlo simulation.

Packaging during the passaging of viruses in cell cultures yields various phenotypes and is regulated by viral protein expression in infected cells. Although such a packaging mechanism has a profound effect in controlling the virus yield, little is known about the underlying statistical models followed by virus packaging and protein expression among cells infected with the virus. A predictive framework combining identification of the probability density function (PDF) based on log-likelihood and using the PDF for Monte-Carlo simulations is developed. The Birnbaum-Saunders distribution was found to be consistent with all three-virus packaging levels, including nucleocapsids/occlusion-derived virus (ODV), ODVs/polyhedra, and polyhedra/cell for both wild-type and genetically modified AcMNPV. Next, it was demonstrated that PDF fitting could be used to compare two viruses having distinctly different genetic configurations. Finally, the identified PDF can be incorporated in RNA synthesis parameters for baculovirus infection to predict the cell-to-cell variability in protein expression using Monte-Carlo simulations. The proposed tool can be used for the estimation of uncertainty in the kinetic parameter and prediction of cell-to-cell variability for other biological systems.

Characterization of a Chrysodeixis includens nucleopolyhedrovirus Isolate from Brazilian Cerrado and Assessment of its Co-Infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus

Abstract Chrysodeixis includens has become the major Lepidopteran pest of soybean crops, especially in the Brazilian Cerrado (savanna) region. A native isolate of Chrysodeixis includens nucleopolyhedrovirus (ChinNPV) from this region, Buritis, MG, was assessed for its biological and molecular features. In addition, in vitro co-infection with Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV), another virus of an important soybean pest, was tested. The ChinNPV-Buritis isolate presented an average LC50 of 7,750 occlusion bodies (OBs)/ml of diet in C. includens larvae. Analysis of restriction endonuclease profiles of viral DNA revealed similarities with previously described ChinNPV isolates IE, IF, and IG from Brazil, although the presence of submolar bands indicates genetic heterogeneity. Optical microscopy analysis in conjunction with quantitative PCR (qPCR) demonstrated in vitro infection of this isolate in IPLB-SF-21AE, Sf9, and BTI-Tn-5B1-4 cell lines, but the amount of ChinNPV tends to decrease through serial passages. The qPCR method developed in this study successfully detected both AgMNPV and ChinNPV from cell culture and from infected larvae. The cell line Tn-5B1-4 is indicated for future development of in vitro production and co-infection studies.

Baculovirus-challenge and poor nutrition inflict within-generation fitness costs without triggering transgenerational immune priming.

Invertebrate hosts that survive pathogen challenge can produce offspring that are more resistant to the same pathogen via immune priming, thereby improving the fitness of their offspring in the same pathogen environment. Most evidence for immune priming comes from exposure to bacteria and there are limited data on other groups of pathogens. Poor parental nutrition has also been shown to result in the transgenerational transfer of pathogen resistance and increased immunocompetence. Here, we combine exposure to an insect DNA virus with a change in the parental diet to examine both parental costs and transgenerational immune priming. We challenged the cabbage looper, Trichoplusia ni, with a low dose of the baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and altered dietary protein to carbohydrate ratio (p:c ratio) after virus exposure. Insects fed a low protein diet had lower haemolymph protein concentrations, and exhibited costs of smaller pupae and slower development, while survivors of virus challenge developed more slowly, irrespective of p:c ratio, and those that were virus-challenged and fed on a low protein diet showed a reduction in haemocyte density. In addition, AcMNPV-challenged parents laid fewer eggs earlier in egg laying although egg size was the same as for unchallenged parents. There was no evidence for increased resistance to AcMNPV (immune priming) or changes in haemocyte number (as proxy for constitutive cellular immunity) in the offspring either as a result of parental AcMNPV-challenge or low dietary p:c ratio. Therefore, although pathogen-challenge and nutritional changes can affect host development and reproduction, this does not necessarily translate into transgenerational immune priming. Our findings contrast with an earlier study on another type of baculovirus, a granulovirus, where immune priming was suggested. This indicates that transgenerational immune priming is not universal in invertebrates and is likely to depend on the host-pathogen system, or the level of pathogen exposure and the type of dietary manipulation. Identifying whether immune priming or transgenerational effects are relevant in field populations, remains a challenge.

Phenotypic Variation in Overwinter Environmental Transmission of a Baculovirus and the Cost of Virulence.

A pathogen's ability to persist in the environment is an ecologically important trait, and variation in this trait may promote coexistence of different pathogen strains. We asked whether naturally occurring isolates of the baculovirus that infects gypsy moth larvae varied in their overwinter environmental transmission and whether this variation was consistent with a trade-off or an upper limit to virulence that might promote pathogen diversity. We used experimental manipulations to replicate the natural overwinter infection process, using 16 field-collected isolates. Virus isolates varied substantially in the fraction of larvae infected, leading to differences in overwinter transmission rates. Furthermore, isolates that killed more larvae also had higher rates of early larval death in which no infectious particles were produced, consistent with a cost of high virulence. Our results thus support the existence of a cost that could impose an upper limit to virulence even in a highly virulent pathogen.

Field testing Chinese and Japanese gypsy moth nucleopolyhedrovirus and disparvirus against a Chinese population of Lymantria dispar asiatica in Huhhot, Inner Mongolia, People's Republic of China.

The activity of three geographic isolates of the gypsy moth nucleopolyhedrovirus (LdMNPV) was evaluated in field trials against larvae of the Chinese population of Lymantria dispar asiatica Vnukovskij in Inner Mongolia, People's Republic of China, in 2004, 2005, and 2006. Although the Chinese isolate of the virus, LdMNPV-H, was the most pathogenic of the isolates tested, having the lowest mean lethal concentration causing 50% and 95% larval mortality, the increase in efficacy that would be obtained by incorporating this isolate into a commercial product does not justify the time or expense required to register it for use in the United States or Canada. The commercially available North American isolate, LdMNPV-D, was moderately pathogenic, whereas the Japanese isolate, LdMNPV-J, was the least pathogenic. The slopes of the dose-response regression lines for the three virus isolates indicated that the Chinese gypsy moth larvae were more homogenously susceptible to LdMNPV-H and LdMNPV-D than to LdMNPV-J. Time-response data showed that LdMNPV-J was significantly more virulent, but at a much higher dose, than the other two isolates, causing 50% mortality in the shortest time, followed by LdMNPV-H and LdMNPV-D. Rainfall immediately after the application of LdMNPV-D in 2005 resulted in significantly reduced gypsy moth larval mortality.

A simplified low-cost diet for rearing Spodoptera exigua (Lepidoptera: Noctuidae) and its effect on S. exigua nucleopolyhedrovirus production.

A low-cost simplified diet has been successfully developed for rearing Spodoptera exigua larvae under laboratory conditions. The cost of ingredients was lower than that of the standard diet based on a modified tobacco hornworm, Manduca sexta (L.), diet. The simplified diet fulfilled larval nutritional requirements without apparent adverse effects on the reproductive capacity of the insect. Survival, pupal sex ratio, and fecundity registered in insects that were reared on the simplified diet did not differ from those observed on the standard diet. The mean larval development period of insects that consumed the simplified diet was also similar to that of insects that consumed the standard diet, whereas weight of pupae and adult longevity were significantly higher in insects reared on simplified diet. Larvae consumed approximately 11% more of the standard diet compared with the simplified diet and a corresponding increase was observed in the number of larvae that could be reared through to pupation on each liter of simplified diet. The production of S. exigua multiple nucleopolyhedrovirus (SeMNPV) occlusion bodies (OBs) in insects grown on each type of diet was also evaluated. Weights of larvae at inoculation and at death, OB yields and biological activity of OBs did not differ significantly for each type of diet. The simplified low-cost diet appears suitable for large-scale in vivo production of SeMNPV OBs.

Optimal control of gypsy moth populations.

This study investigates an optimal strategy for the cost effective control of gypsy moth populations. Gypsy moth populations cycle between low sparse numbers to high outbreak levels and it is during the outbreak levels that the moths cause extensive damage to plant foliage which can lead to deforestation. Deforestation can result in significant economic damage to infested areas, and consequently, there have been many efforts to control moth populations. One effective method of control is the use of the biocontrol agent, Gypchek, but its production is costly. We develop a mathematical model which combines population dynamics and optimal control of the moth population to explore strategies by which the total cost of the gypsy moth problem (economic damage and cost of Gypchek) can be minimized.

Flexible diet choice offsets protein costs of pathogen resistance in a caterpillar.

Mounting effective resistance against pathogens is costly in terms of energy and nutrients. However, it remains unexplored whether hosts can offset such costs by adjusting their dietary intake so as to recoup the specific resources involved. We test this possibility by experimentally challenging caterpillars (Spodoptera littoralis) with a highly virulent entomopathogen (nucleopolyhedrovirus), under dietary regimes varying in the content of protein and digestible carbohydrate. We found that dietary protein influenced both resistance to pathogen attack and constitutive immune function to a greater extent than did dietary carbohydrate, indicating higher protein costs of resistance than energy costs. Moreover, when allowed to self-compose their diet, insects surviving viral challenge increased their relative intake of protein compared with controls and those larvae dying of infection, thus demonstrating compensation for protein costs associated with resistance. These results suggest that the change in the host's nutritional demands to fight infection induces a compensatory shift in feeding behaviour.

The mosaic structure of the polyhedrin gene of the Autographa californica nucleopolyhedrovirus (AcMNPV).

The polyhedrin (polh) gene of nucleopolyhedroviruses (NPVs) encodes for the matrix protein of the virus occlusion body and is one of the most conserved baculovirus genes. Previous analyses of different NPV genes and polh genes provided conflicting results indicating that the Autographa californica nucleopolyhedrovirus (AcMNPV) is generally a member of the so-called group I NPVs and is most closely related to Rachiplusia ou (Ro) NPV, whereas the AcMNPV polh is more similar to the polh of the group II NPVs. A comparative analysis of the AcMNPV polh and its closest neighbours within group I and group II NPV, the RoMNPV and the Thysanoplusia orichalcea (Thor) NPV, was performed using Hidden Markov Models for detecting recombination. The result provided strong evidence that the AcMNPV polh is a chimerical gene which consists of a mosaic of group I and group II NPV specific sequences.

Assessment of foreign protein production by recombinant Heliothis (Helicoverpa) armigera entomopoxviruses in Spodoptera frugiperda cells.

This report describes the first production of recombinant forms of Heliothis (Helicoverpa) armigera entomopoxvirus (HaEPV). These HaEPVs are engineered at either the spheroidin or fusolin locus, to produce the green fluorescent marker protein (GFP). The growth properties of these recombinant HaEPVs, in comparison to the parental HaEPV, were assessed in cultured Spodoptera frugiperda Sf9 cells. Additionally, GFP production by these recombinant HaEPVs was compared to that of a GFP-expressing recombinant of the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) in the same in vitro system, at various multiplicities of infection. Expression of GFP from the HaEPV spheroidin locus produced up to 60% of that generated from the AcNPV polyhedrin locus, albeit over a longer period of infection. A considerably lower yield was recorded from the HaEPV fusolin locus, a result that contrasted markedly with the apparent activity of this promoter in caterpillar infections in vivo. The potential applications for further development of HaEPV expression systems are discussed.