BUSCADOR RECOLECTA

A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus
(SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac).
Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic
DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective
when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than
those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or
budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null
compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity)
to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that
SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion,
resulting in increased numbers of nucleocapsids within ODVs., This study received financial support from the Spanish Ministry for Science and Technology (AGL2008-05456-C03-01, AGL2011-30352-C02-01). I.B. received a CSIC studentship.

With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it
becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential
to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua
multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected
transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected
insects: i) healthy males (H=)6healthy females (HR); ii) infected males (I=)6healthy females (HR); iii) healthy males
(H=)6infected females (IR) and iv) infected males (I=)6infected females (IR). Experimental adults and their offspring were
analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to
the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Malemediated
venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination
studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female
offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virusbased
insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between
bioinsecticide applications., This work was supported by the Spanish Ministry for Science and Technology, project AGL2011-30352-C02-01. CV received a predoctoral fellowship
from the Universidad Pública de Navarra.

Naturally occurring covert infections in lepidopteran populations can involve multiple viruses with potentially different transmission strategies. In this study, we characterized covert infection by two RNA viruses, Spodoptera exigua iflavirus 1 (SeIV-1) and Spodoptera exigua iflavirus 2 (SeIV-2) (family Iflaviridae) that naturally infect populations of Spodoptera exigua, and examined their influence on susceptibility to patent disease by the nucleopolyhedrovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) (family Baculoviridae). The abundance of SeIV-1 genomes increased up to ten-thousand-fold across insect developmental stages after surface contamination of host eggs with a mixture of SeIV-1 and SeIV-2 particles, whereas the abundance of SeIV-2 remained constant across all developmental stages. Low levels of SeIV-2 infection were detected in all groups of insects, including those that hatched from surface-decontaminated egg masses. SeIV-1 infection resulted in reduced larval weight gain, and an unbalanced sex ratio, whereas larval developmental time, pupal weight, and adult emergence and fecundity were not significantly affected in infected adults. The inoculation of S. exigua egg masses with iflavirus, followed by a subsequent infection with SeMNPV, resulted in an additive effect on larval mortality. The 50% lethal concentration (LC50) of SeMNPV was reduced nearly 4-fold and the mean time to death was faster by 12 h in iflavirus-treated insects. These results suggest that inapparent iflavirus infections may be able to modulate the host response to a new pathogen, a finding that has particular relevance to the use of SeMNPV as the basis for biological pest control products., This research was funded by Spanish Ministry of Science and Innovation (AGL2011-30352-C02-01). AC received a doctoral grant from AGL2011-30352-C02-01.

Nucleopolyhedroviruses (NPV, Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems., This review was funded by the Ministerio de Economía y Competitividad, Spain, project number AGL2017-83498-C2-1-R and previous projects AGL2014-57752-C2-1-R, AGL2011-30352-CO2-01, AGL2008-05456-C03-01, AGL2005-07909-CO3-01, and AGL2002-04320-C02-01.

Superinfection exclusion is the ability of an established virus to interfere with a second virus infection. This effect was studied in
vitro during lepidopteran-specific nucleopolyhedrovirus (genus Alphabaculovirus, family Baculoviridae) infection. Homologous
interference was detected in Sf9 cells sequentially infected with two genotypes of Autographa californica multiple nucleopolyhedrovirus
(AcMNPV), each one expressing a different fluorescent protein. This was a progressive process in which a sharp decrease
in the signs of infection caused by the second virus was observed, affecting not only the number of coinfected cells observed,
but also the level of protein expression due to the second virus infection. Superinfection exclusion was concurrent with
reorganization of cytoplasmic actin to F-actin in the nucleus, followed by budded virus production (16 to 20 h postinfection).
Disruption of actin filaments by cell treatment with cytochalasin D resulted in a successful second infection. Protection against
heterologous nucleopolyhedrovirus infection was also demonstrated, as productive infection of Sf9 cells by Spodoptera frugiperda
nucleopolyhedrovirus (SfMNPV) was inhibited by prior infection with AcMNPV, and vice versa. Finally, coinfected cells
were observed following inoculation with mixtures of these two phylogenetically distant nucleopolyhedroviruses—AcMNPV
and SfMNPV—but at a frequency lower than predicted, suggesting interspecific virus interference during infection or replication.
The temporal window of infection is likely necessary to maintain genotypic diversity that favors virus survival but also permits
dual infection by heterospecific alphabaculoviruses., I.B. received a CSIC studentship. This research was supported by the
project AGL2011-30352-CO2-01 (Universidad Pública de Navarra, Pamplona,
Spain) and Oxford Brookes University, Oxford, United Kingdom.

Virus transmission and the prevalence of infection depend on multiple factors, including
the interaction with other viral pathogens infecting the same host. In this study,
active replication of an iflavirus, Spodoptera exigua iflavirus 1 (order Picornavirales)
was observed in the offspring of insects that survived following inoculation with a
pathogenic baculovirus, Spodoptera exigua multiple nucleopolyhedrovirus. Tracking
the origin of the iflavirus suggested the association of this virus with the occlusion
bodies of the baculovirus. Here we investigated the effect of this association on the
stability and infectivity of both viruses. A reduction in baculovirus pathogenicity,
without affecting its infectivity and productivity, was observed when associated with
the iflavirus. In contrast, viral association increased the infectivity of the iflavirus and
its resistance to ultraviolet radiation and high temperature, two of the main factors
affecting virus stability in the field. In addition, electron microscopy analysis revealed
the presence of particles resembling iflavirus virions inside the occlusion bodies of the
baculovirus, suggesting the possible co-occlusion of both viruses. Results reported here
are indicative of facultative phoresis of a virus and suggest that virus virus interactions
may be more common than currently recognized, and may be influential in the ecology
of baculovirus and host populations and in consequence in the use of baculoviruses as
biological insecticides., This study received financial support from the Spanish Ministry for Science and Technology (AGL2011-30352-C02 and AGL2014-57752-C2).

Biological insecticides based on Spodoptera exigua multiple nucleopolyhedrovirus
(SeMNPV) can efficiently control S. exigua larvae on field and greenhouse crops in many
parts of the world. Spanish wild populations and laboratory colonies of S. exigua are infected
by two iflaviruses (SeIV-1 and SeIV-2). Here we evaluated the effect of iflavirus co-infection
on the insecticidal characteristics of SeMNPV occlusion bodies (OBs). Overall, iflavirus coinoculation
consistently reduced median lethal concentrations (LC50) for SeMNPV OBs
compared to larvae infected with SeMNPV alone. However, the speed of kill of SeMNPV
was similar in the presence or absence of the iflaviruses. A reduction of the weight gain
(27%) associated with iflavirus infection resulted in a 30% reduction in total OB production
per larva. Adult survivors of SeMNPV OB inoculation were examined for covert infection.
SeMNPV DNA was found to be present at a high prevalence in all SeIV-1 and SeIV-2 coinfection
treatments. Interestingly, co-inoculation of SeMNPV with SeIV-2 alone or in mixtures
with SeIV-1 resulted in a significant increase in the SeMNPV load of sublethally
infected adults, suggesting a role for SeIV-2 in vertical transmission or reactivation of sublethal
SeMNPV infections. In conclusion, iflaviruses are not desirable in insect colonies used
for large scale baculovirus production, as they may result in diminished larval growth,
reduced OB production and, depending on their host-range, potential risks to non-target
Lepidoptera., This study was funded by the Spanish Ministerio de Ciencia e Innovación, project number AGL2011-30352-C02-01. AC received a student scholarship from the Ministerio de Economía y Competitividad.