Biological and genetic characterization of a Pakistani isolate of <i>Spodoptera litura</i> nucleopolyhedrovirus

<p><i>Spodoptera litura</i> is an emerging insect pest in a wide range of crops worldwide. The insect is difficult to control because of resistance development to synthetic insecticides and emerging resistance to <i>Bacillus thuringiensis</i> toxins. Therefore, there is a need to develop biological control agents, preferably from an indigenous source to avoid risks associated with the importation of exotic natural antagonists. A Pakistani isolate of <i>S. litura</i> nucleopolyhedrovirus (SpltNPV, <i>Baculoviridae</i>), SpltNPV-Pak-BNG, was obtained from the field and characterized biologically and genetically, and compared to a SpltNPV reference isolate, SpltNPV-G1, thought to be of Chinese origin. The dose–mortality response (LD₅₀) of SpltNPV-Pak-BNG was not significantly different from that of the reference isolate SpltNPV-G1, but the time-to-death (LT₅₀) was significantly shorter for SpltNPV-Pak-BNG than for SpltNPV-G1. DNA restriction enzyme profiling indicated that SpltNPV-Pak-BNG and SpltNPV-G1 are different viruses. Sequence analysis of ‘ORF24’, specific for SpltNPV (and S. littoralis NPV as ORF21), and the conserved baculovirus core genes <i>polyhedrin</i>, <i>DNApol</i>, <i>pif-2</i> and <i>lef-8</i> confirmed that this was indeed the case and that SpltNPV-Pak-BNG is a genuine SpltNPV variant, whereas the SpltNPV-G1 isolate we used is, in fact, a SpliNPV variant, renamed to SpliNPV-G1. The newly isolated SpltNPV-Pak-BNG has the potential for development as a biocontrol agent of <i>S. litura</i> in Pakistan.</p

Frequency of mixed-genotype infection at different doses.

<p>The number of mixed-genotype infected larvae and the total number of larvae tested by qPCR (mixed/total), observed frequency of mixed-genotype infection (<i>f</i>(A∩B)), IAH predicted frequency of mixed-genotype infection (<i>P'</i>(A∩B)), and the significance level of a one-sided binomial test comparing <i>f</i>(A∩B) and <i>P'</i>(A∩B) (significance) are given. * indicates statistical significance at a 0.05 significance threshold, ** at a 0.01 threshold, and *** at a 0.001 threshold.</p

The effects of heterogeneity in host susceptibility.

<p>We assume a host population that is composed of 1, 2, 4 or 6 classes of individuals, varying in their susceptibility to a virus. The number of classes is given in the legend, and applies to all three panels. (E.g. A value of ‘1’ indicates the infection probabilities are the same for all hosts, so in this instance the host population is homogeneous. ‘6’ indicates that there are six host classes with different susceptibilities to the virus) The following infection probabilities were assumed: 1 class, 10^−4.5; 2 classes, 10⁻⁴, 10⁻⁵; 4 classes, 10⁻³, 10⁻⁴; 10⁻⁵, 10⁻⁶; 6 classes, 10⁻², 10⁻³, 10⁻⁴; 10⁻⁵, 10⁻⁶, 10⁻⁷. The geometric mean infection probability is 10^−4.5 in all four cases. A virus population composed of two genotypes in a 1∶1 mixture, and no differences in infection probability for these genotypes, was assumed. In Panel A, the dose response relationship is illustrated. On the x-axis is the log of dose, and on the y-axis mortality. Note that as more host classes are introduced, the dose response relationship becomes shallower. In Panel B is the frequency of mixed-genotype infection, which follows a similar trend with dose. Panel C is the relationship between host mortality (x-axis) and the frequency of mixed-genotype infection (y-axis). The solid line is a 1∶1 relationship between mortality and the frequency of mixed-genotype infection. As a more heterogeneity is introduced in the host population, the frequency of mixed-genotype infection becomes higher and eventually approaches the 1∶1 line (both micro-parasite genotypes are established in all hosts at any level of mortality).</p

Dose response for AcMNPV infection of <i>S. exigua</i> L5.

<p>On the x-axis is the log10 of the virus dose (OBs per ml) droplet fed to larvae. The proportion of hosts dying is on the y-axis. The different symbols represent different replicates, which covered different ranges in dose. The line represents the IAH dose response relationship by non-linear regression (mortality  = 1 - exp(-<i>p</i>· OB concentration), SPSS 15.0), rendering <i>p</i> = 2.205×10⁻⁷.</p

Ratio of genotype A to genotype B in infected hosts.

<p>The log-transformed genotype ratio (A:B) is given for <i>S. exigua</i> L5 larvae at different inoculum OB concentrations, as indicated in the upper left-hand corner of each panel. On the x-axis is the log10 of the genotype ratio (A:B), and on the y-axis frequency. Host survival <i>(S)</i> decreased with inoculum OB concentration, from <i>S</i> = 0.72 (10⁵ OBs per ml) to <i>S</i> = 0.03 (10⁸ OBs per ml). The number of hosts per dose are 14 (10⁵ and 10⁶ OBs per ml), 12 (10⁷ OBs per ml) and 8 (10⁸ OBs per ml).</p

The predicted distribution of host susceptibility.

<p>The distribution of host susceptibility predicted by model 2 with fitted parameters: <i>p</i> = 0.0059, <i>α</i> = 0.335, <i>β</i> = 143 (<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002097#pcbi-1002097-t003" target="_blank">Table 3</a>). Host susceptibility is characterized by the overall probability of disease causation for a single ODV, <i>pξ</i>, which is the product of the <i>per virion</i> chances of successful penetration, <i>p</i>, and the probability of successful infection given successful penetration, <i>ξ</i>. The shape of the cumulative distribution of <i>pξ</i> is the same as that of the beta distribution for <i>ξ</i>, but the domain of the former is limited to (0, <i>p</i>], and the distribution is accordingly shifted to the left compared to the beta distribution for <i>ξ w</i>hich has (0,1] as its domain. A logarithmic scale is used for the abscissa to represent the broad range of susceptibility in the host population, which is reflected in shallow dose response and is – as shown in this paper – intrinsically associated with a high incidence of mixed genotypes in cadavers.</p

Fitted models and experimental data.

<p>Fitted Models 1–4 compared with experimental data. In all panels, the log of the dose is on the x-axis and frequency on the y-axis. Dose mortality responses are given in the left-hand panels (A–D). Here, diamonds are the experimental data and the lines model predictions. Relationships between dose and rate of mixed-genotype infection in cadavers are presented in the right hand panels (E–H). Again, markers denote experimental data while lines denote model predictions: red circles and the red solid line denote mixed infection with genotypes A and B, green triangles and the green dotted line denote infections with genotype A only, and blue squares and the fine dotted blue line denote infections with genotypes B only. Horizontally adjacent panels pertain to the same model, as indicated in the left hand panel (e.g., panels A and E correspond to model 1). Model 2 gives the best description of the data (see <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1002097#pcbi-1002097-t003" target="_blank">Table 3</a>).</p

Symbols that pertain throughout the paper.

<p>Symbols that pertain throughout the paper.</p

An overview of the four models postulated.

<p>An overview of the four models postulated.</p

Model selection among variants of the <i>Fokker-Planck</i> diffusion model describing beetle dispersal in the field experiment.

<p>A single parameter value is shown when habitats or trap types were not distinguished in a model variant. The value of <i>π</i>₁ is 1 throughout. If no value for <i>π</i>₂ is shown, it was set to 1 and not included in the calibration. The negative log-likelihood (NLL) is a measure of the goodness of fit of the model to the data. ΔAIC is the difference in Akaike's information criterion between a model variant and the model variant with most support of the data (model 1). Models variants of which the ΔAIC is smaller than two are considered equivalent and have equal support from the data.</p><p><i>μ</i>₁: motility in oilseed radish; <i>μ</i>₂: motility in rye; <i>ξ</i>: relative loss rate due to removal other than recapture (e.g. mark wear and mortality); <i>ω</i>₀: trap-coefficient for trapping stations without screens; <i>ω</i>₁: trap-coefficient for trapping stations with screens; <i>π</i>₁: multiplication factor of the flux of beetles from oilseed radish to rye; <i>π</i>₂: multiplication factor of the flux of beetles from rye to oilseed radish.</p><p>Model selection among variants of the <i>Fokker-Planck</i> diffusion model describing beetle dispersal in the field experiment.</p