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The aim of this study, conducted in 2018 and 2020, was to investigate the bioactivity of a local baculovirus isolate, Hyphantria cunea granulovirus (HycuGV), in seven lepidopteran pests. Based on data collected 10 days after exposure, HycuGV was found to infect Malacosoma neustria (L., 1758) (Lepidoptera: Lasiocampidae), Lymantria dispar (L., 1758) (Lepidoptera: Erebidae), Helicoverpa armigera (Hubner, 1805) (Lepidoptera: Noctuidae) and Spodoptera exigua (Hubner, 1808) (Lepidoptera: Noctuidae) larvae as well as its host Hyphantria cunea (Drury, 1773) (Lepidoptera: Erebidae). However, it did not infect Spodoptera littoralis (Boisduval, 1883) (Lepidoptera: Noctuidae) and Cydia pomonella (L., 1758) (Lepidoptera: Tortricidae). A HycuGV dose rate experiment indicated LC50 of 4.7x10(5) occlusion bodies (OBs)/ml in H. cunea, 5.6x10(6) OBs/ml in L. dispar, 7x10(7) OBs/ml in S. exigua, 1.5x10(9) OBs/ml in M. neustria and 7.7x10(9) OBs/ml in H. armigera. HycuGV was infectious to S. exigua and L. dispar, but only provided effective control in M. neustria and H. armigera at high dose rates. These findings demonstrate that HycuGV can be highly effective for control of S. exigua, L. dispar and H. cunea.

The use of bioproducts developed from active viral ingredients is a promising strategy in integrated pest management. Accordingly, the VG010 betabaculovirus has been identified as an effective agent in controlling the hornworm Erinnyis ello. The development of a wettable powder formulation using VG010 as an active ingredient in a biopesticide must have a guaranteed efficacy. An analysis of the unit operations involved in the formulation was carried out using proper operating conditions and production equipment. The active ingredient and finished product characterization revealed the repeatability of variables among batches. The mixing and drying processes were evaluated using operational parameters such as the mixing index and drying kinetics. From this analysis, the most favorable mixing time and drying temperature were selected to obtain a homogeneous product and high insecticidal activity. Viral concentrations, pH of the liquid mixtures, and drying temperature were established as control parameters of the critical points.

We described a novel baculovirus isolated from the polyphagous insect pest Rachiplusia nu. The virus presented pyramidal-shaped occlusion bodies (OBs) with singly-embed nucleocapsids and a dose mortality response of 6.9 × 103 OBs/ml to third-instar larvae of R. nu. The virus genome is 128,587 bp long with a G + C content of 37.9% and 134 predicted ORFs. The virus is an alphabaculovirus closely related to Trichoplusia ni single nucleopolyhedrovirus, Chrysodeixis chalcites nucleopolyhedrovirus, and Chrysodeixis includens single nucleopolyhedrovirus and may constitute a new species. Surprisingly, we found co-evolution among the related viruses and their hosts at species level. Besides, auxiliary genes with homologs in other baculoviruses were found, e.g. a CPD-photolyase. The gene seemed to be result of a single event of horizontal transfer from lepidopterans to alphabaculovirus, followed by a transference from alpha to betabaculovirus. The predicted protein appears to be an active enzyme that ensures likely DNA protection from sunlight.

Incluye material complementario Enhancins are metalloproteinases that facilitate baculovirus infection in the insect midgut. They are more prevalent in granuloviruses (GVs), constituting up to 5% of the proteins of viral occlusion bodies (OBs). In nucleopolyhedroviruses (NPVs), in contrast, they are present in the envelope of the occlusion-derived virions (ODV). In the present study, we constructed a recombinant Autographa californica NPV (AcMNPV) that expressed the Trichoplusia ni GV (TnGV) enhancin 3 (En3), with the aim of increasing the presence of enhancin in the OBs or ODVs. En3 was successfully produced but did not localize to the OBs or the ODVs and accumulated in the soluble fraction of infected cells. As a result, increased OB pathogenicity was observed when OBs were administered in mixtures with the soluble fraction of infected cells. The mixture of OBs and the soluble fraction of Sf9 cells infected with BacPhEn3 recombinant virus was ~3- and ~4.7-fold more pathogenic than BacPh control OBs in the second and fourth instars of Spodoptera exigua, respectively. In contrast, when purified, recombinant BacPhEn3 OBs were as pathogenic as control BacPh OBs. The expression of En3 in the soluble fraction of insect cells may find applications in the development of virus-based insecticides with increased efficacy. This study received financial support from the Spanish Ministry project AGL2017-83498-C2-1-R. Bioinsectis SL funded the activities of ABF.

Purified occlusion bodies (OBs) of Mythimna (formerly Pseudaletia) unipuncta (the true armyworm) granulovirus Hawaiian strain (MyunGV-A) were observed, showing typical GV morphological characteristics under scanning and transmission electron microscopy (EM). The genome of MyunGV-A was completely sequenced and analysed. The genome is 176,677 bp in size, with a G+C content of 39.79%. It contains 183 open reading frames (ORFs) encoding 50 or more amino acids with minimal overlap. Comparison of MyunGV-A with TnGV, XcGV, and HearGV genomes revealed extensive sequence similarity and collinearity, and the four genomes contain the same nine homologous regions (hrs) with conserved structures and locations. Three unique genes, 12 baculovirus repeated ORF (bro), 2 helicase, and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome, in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. In addition, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV (99% amino acid identity) according to a phylogenetic tree based on the combined amino acid sequences of 38 core gene contents.

The Cydia pomonella granulovirus (CpGV) has been used as a biological control agent of codling moth (Cydia pomonella), a severe global pest on pome fruit. Despite the economic importance, our knowledge of its molecular biology is still limited and a detailed picture of its gene expression is still missing. Here, we sequenced the transcriptome of codling moth larvae infected with the Mexican isolate CpGV-M and analyzed the expression of viral genes at 12, 48, and 96 h post infection (hpi). The results showed that two genes (p6.9 and pp31/39K) related to DNA binding of virus production, were highly expressed at 48 and 96 hpi. From 48 to 96 hpi, the expression of genes associated with virus replication and dissemination decreased, whereas the expression of genes related to infectious virion production and per os infectivity increased. This study provides a comprehensive view of CpGV gene expression patterns in host larvae.

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonellagranulovirus (CpGV) and Cryptophlebia leucotretagranulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

The cassava hornworm Erinnyis ello ello (Lepidoptera: Sphingidae) is an important pest in Brazil. This insect feeds on host plants of several species, especially Manihot esculenta (cassava) and Hevia brasiliensis (rubber tree). Cassava hornworm outbreaks are quite common in Brazil and can cause great impact over crop production. Granulare and polyhedral-shaped occlusion bodies (OBs) were observed in extracts of dead E. ello larvae from rubber-tree plantations by light and scanning electron microscopy (SEM), suggesting a mixed infection. The polyhedral-shaped OB surface revealed indentations that resemble those found in cypovirus polyhedra. After OB nucleic acid extraction followed by cDNA production and Illumina deep-sequencing analysis, the results confirmed for the presence of a putative novel cypovirus that carries ten segments and also a betabaculovirus (Erinnyis ello granulovirus, ErelGV). Phylogenetic analysis of the predicted segment 1-enconded RdRP showed that the new cypovirus isolate is closely related to a member of species Cypovirus 2, which was isolated from Inachis io (Lepidoptera: Nymphalidae). Therefore, we named this new isolate Erinnyis ello cypovirus 2 (ErelCPV-2). Genome in silico analyses showed that ErelCPV-2 segment 8 (S8) has a predicted amino acid identity of 35.82 % to a hypothetical protein of betabaculoviruses. This putative protein has a cGAMP-specific nuclease domain related to the poxvirus immune nucleases (poxins) from the 2′,3′-cGAMP-degrading enzyme family.

Background The hornworn Erinnyis ello is the major pest of natural rubber crops in Colombia, mainly controlled using toxic chemical insecticides. The use of E. ello Betabaculovirus is an environmentally sustainable alternative for its control. The aim of the present work was to characterize a prototype biopesticide formulation and evaluate its efficacy under different conditions. Results Quality control evaluations of formulated biopesticide revealed that all the parameters evaluated were under the permissible level. The lethal concentrations LC50 and LC90 of the biopesticide were 4.3 × 103 and 5.5 × 104 occlusion bodies (OBs) mL-1 , respectively. Biopesticide efficacies against second and fourth instar larvae under greenhouse conditions were higher than 80%. Evaluation of two application rates in a clonal garden resulted in 84% and 88% efficacy, comparable to that obtained with the chemical. The biopesticide in a commercial plantation showed efficacies between 74% and 82%. Biopesticide post-application persistence was estimated at least in 1 week under field natural conditions. Results allowed selection of the lowest evaluated dose (1 × 1011 OBs ha-1 ) as the basis for further field evaluations. Conclusion Formulated ErelGV showed high efficacy to control the hornworm in rubber crops and high potential to be included in integrated pest management programs, thus it could be an interesting alternative to replace agrochemicals. © 2018 Society of Chemical Industry.

A broad survey of the fall webworm (Lepidoptera: Erebidae) populations in agricultural and forested areas in the Central Black Sea Region of Turkey led to the detection of a granulovirus (G...

BackgroundErinnyis ello granulovirus(ErelGV) is a betabaculovirus infecting caterpillars of the sphingid mothE. ello ello(cassava hornworm), an important pest of cassava crops (Manihot esculenta). In this study, the genome of seven field isolates of the virus ErelGV were deep sequenced and their inter-and intrapopulational sequence diversity were analyzed.ResultsNo events of gene gain/loss or translocations were observed, and indels were mainly found within highly repetitive regions (direct repeats,drs). A naturally occurring isolate from Northern Brazil (Acre State, an Amazonian region) has shown to be the most diverse population, with a unique pattern of polymorphisms. Overall, non-synonymous substitutions were found all over the seven genomes, with no specific gathering of mutations on hotspot regions. Independently of their sizes, some ORFs have shown higher levels of non-synonymous changes than others. Non-core genes of known functions and structural genes were among the most diverse ones; and as expected, core genes were the least variable genes. We observed remarkable differences on diversity of paralogous genes, as in multiple copies ofp10, fgf, andpep. Another important contrast on sequence diversity was found on genes encoding complex subunits and/or involved in the same biological processes, aslate expression factors(lefs) andper os infectivity factors(pifs). Interestingly, several polymorphisms in coding regions lie on sequences encoding specific protein domains.ConclusionsBy comparing and integrating information about inter-and intrapopulational diversity of viral isolates, we provide a detailed description on how evolution operates on field isolates of a betabaculovirus. Our results revealed that 35-41% of the SNPs of ErelGV lead to amino acid changes (non-synonymous substitutions). Some genes, especially non-core genes of unknown functions, tend to accumulate more mutations, while core genes evolve slowly and are more conserved. Additional studies would be necessary to understand the actual effects of such gene variations on viral infection and fitness.

Current knowledge of the field resistance of codling moth (CM, Cydia pomonella, L) against Cydia pomonella granulovirus (CpGV) is based mainly on the interaction between the Mexican isolate CpGV-M and CpRR1, a genetically homogeneous CM inbreed line carrying type I resistance. The resistance level of laboratory-reared CpRR1 to CpGV-M was recently found to have decreased considerably, compared to the initially high resistance. To understand the background of this phenomenon, CpRR1 larvae were exposed over several generations to CpGV-M for re-selection of the original resistance level. After five and seven generations of selection, new CpRR1_F5 and CpRR1_F7 lines were established. The resistance ratio of these selected lines was determined by full range bioassays. The CpRR1_F5 strain regained a higher level of resistance against CpGV up to 104-fold based on LC50 values compared to susceptible larvae (CpS), which indicated that the absence of virus selection had resulted in a reduction of resistance under laboratory rearing conditions. In addition, some fitness costs of fecundity were observed in CpRR1_F5. Single-pair crossings between CpRR1_F5 or CpRR1_F7 with susceptible CpS moths revealed a dominant but not fully sex-linked inheritance, which suggests a partial loss of previous resistance traits in CpRR1.

Spodoptera exempta nucleopolyhedrovirus (SpexNPV) is a viral pathogen of the African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), a significant agricultural pest of cereal crops in Africa. SpexNPV has been evaluated as a potential insecticide for control of this pest and has served as the subject of research on baculovirus pathology and transmission. Occlusion bodies (OBs) of SpexNPV isolate 244.1 were examined, and the nucleotide sequence of the genome was determined and characterized. SpexNPV-244.1 OBs consisted of irregular polyhedra with a size and appearance typical for alphabaculoviruses. Virions within the polyhedra contained 1-8 nucleocapsids per unit envelope. The SpexNPV-244.1 genome was comprised of a 129,528 bp circular sequence, in which 139 ORFs were annotated. Five homologous regions (hrs) consisting of a variable number of 28-bp imperfect palindromes were identified in the genome. The genome sequence contained the 38 core genes of family Baculoviridae, as well as three ORFs unique to the SpexNPV sequence and one ORF that was apparently acquired by horizontal gene transfer with a betabaculovirus ancestor. Phylogenetic inference with core gene amino acid sequence alignments placed SpexNPV-244.1 in a lineage containing alphabaculoviruses of Spodoptera frugiperda and Spodopotera exigua which in turn is part of a larger group of alphabaculoviruses from the subfamily Noctuinae in the lepidopteran family Noctuidae. Kimura-2-parameter pairwise nucleotide distances indicated that SpexNPV-244.1 represented a different and previously unlisted species in the genus Alphabaculovirus. Gene parity plots indicated that the gene order of SpexNPV-244.l was extensively collinear with that of Spodoptera exigua NPV (SeMNPV). These plots also revealed a group of 17 core genes whose order was conserved in other alpha- and betabaculoviruses.

Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins. IMPORTANCE Baculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

The complete genomes of two novel South African betabaculovirus isolates, namely Phthorimaea operculella granulovirus (PhopGV-SA) and Plutella xylostella granulovirus (PlxyGV-SA), were sequenced and compared to the respective reference isolates PhopGV-1346 and PlxyGV-K1. For both isolates, the genome size and guanine-cytosine (GC) content were similar to those of the respective reference genomes. However, numerous-single nucleotide polymorphisms (SNPs) and several insertions/deletions were observed, revealing the novelty of the isolates. Focus was placed on analysing the observed insertion/deletion events by conducting amino acid sequence alignments for all ORFs of each isolate against all respective ORFs in the corresponding reference isolate. Certain ORFs in each granulovirus genome contained significant insertion/deletion events. In addition, the PlxyGV-SA genome had single-nucleotide insertions/deletions in ORFs 38 and 49 that resulted in the extension and complete overlap of these two ORFs with the neighbouring ORFs 39 and 48, respectively. These novel isolates have significant potential for development and application as biopesticides in South Africa, and the genetic variations observed may have important implications for the biological activity and management of host resistance in the field.

Mutations shape synonymous codon usage bias in certain organism genomes, while selection shapes it in others. Lepidopteran-specific Alphabaculovirus and Betabaculovirus are two large genera in the family of Baculoviridae. In this study, we analyzed the codon usage patterns in 17 baculoviruses, including 10 alphabaculoviruses and 7 betabaculoviruses, which were isolated from seven insect species, and we characterized the codon usage patterns between Alphabaculovirus and Betabaculovirus. Our results show that all the baculoviruses possessed a general weak trend of codon bias. The differences of ENc (effective number of codons) values, nucleotide contents and the impacts of nucleotide content on ENc value within alpha-/betabaculovirus pairs were independent of whether the host species are the same or different. Furthermore, the majority of amino acid sequences adopted codons unequally in all viruses, but the numbers of common preferred codons between alpha- and betabaculoviruses hosted by the same insect species were not significantly different from the differences observed between alpha- and betabaculoviruses hosted by different insect species. In addition, the amino acids that adopt the same synonymous codon composition between alpha- and betabaculoviruses hosted by the same insect species were statistically as few as those between alpha- and betabaculoviruses hosted by different insect species. Correspondence analysis revealed that no major factors resulted in the codon bias in these baculoviruses, implying multiple minor influential factors exist. Neutrality plot analysis indicated that selection pressure dominated mutations in shaping the codon usage. However, the levels of selection pressure were not significantly different among viruses hosted by the same insect species. We expect that evolution would cause the alpha- and betabaculoviruses hosted by the same insect species to share more patterns, but this effect was not observed., Highlights • The codon usage in Alphabaculovirus and Betabaculovirus is weak biased. • Multiple minor influential factors account for the codon usage. • Selection pressure dominates mutations in shaping the codon usage. • The differences of codon usage within alpha-/betabaculovirus pairs are independent.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog ( disa118 ). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64 -null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

Within family Baculoviridae, members of the Betabaculovirus genus are employed as biocontrol agents against lepidopteran pests, either alone or in combination with selected members of the Alphabaculovirus genus. Epinotia aporema granulovirus (EpapGV) is a fast killing betabaculovirus that infects the bean shoot borer (E. aporema) and is a promising biopesticide. Because occlusion bodies (OBs) play a key role in baculovirus horizontal transmission, we investigated the composition of EpapGV OBs. Using mass spectrometry-based proteomics we could identify 56 proteins that are included in the OBs during the final stages of larval infection. Our data provides experimental validation of several annotated hypothetical coding sequences. Proteogenomic mapping against genomic sequence detected a previously unannotated ac110-like core gene and a putative translation fusion product of ORFs epap48 and epap49. Comparative studies of the proteomes available for the family Baculoviridae highlight the conservation of core gene products as parts of the occluded virion. Two proteins specific for betabaculoviruses (Epap48 and Epap95) are incorporated into OBs. Moreover, quantification based on emPAI values showed that Epap95 is one of the most abundant components of EpapGV OBs., Facultad de Ciencias Exactas, Instituto de Biotecnologia y Biologia Molecular

Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories.

The fall webworm (Hyphantria cunea) impacts a wide variety of crops and cultivated broadleaf plant species. The pest is native to North America, was introduced to Europe and has since spread further as far as central Asia. Despite several attempts to control its distribution, the pest continues to spread causing damage all over the world. A naturally occurring baculovirus, Hyphantria cunea granulovirus (HycuGV-Hc1), isolated from the larvae of H. cunea in Turkey appears to have a potential as microbial control agent against this pest. In this report we describe the complete genome sequence and organization of the granulovirus isolate (HycuGV-Hc1) that infects the larval stages and compare it to other baculovirus genomes. The HycuGV-Hc1 genome is a circular double-stranded DNA of 114,825 bp in size with a nucleotide distribution of 39.3% G + C. Bioinformatics analysis predicted 132 putative open reading frames of (ORFs) ≥ 150 nucleotides. There are 24 ORFs with unknown function. Seven homologous repeated regions (hrs) and two bro genes (bro-1 and bro-2) were identified in the genome. Comparison to other baculovirus genomes, HycuGV-Hc1 revealed some differences in gene content and organization. Gene parity plots and phylogenetics confirmed that HycuGV-Hc1 is a Betabaculovirus and is closely related to Plutella xylostella granulovirus. This study expands our knowledge on the genetic variation of HycuGV isolates and provides further novel knowledge on the nature of granuloviruses.

An antagonistic effect of a microsporidium (Nosema sp.) infection on the virulence of Phthorimaea operculella granulovirus (PhopGV) was recorded in potato tuber moth (Phthorimaea operculella) larvae with mixed infections. When the P. operculella colony was infected at a high rate (42.8–100%) with the microsporidium, it was less susceptible to the isolate PhopGV-GR1.1. A virus concentration 1.89 × 105 higher was necessary to cause the same level of mortality produced in the P. operculella colony when it was uninfected or had a low level of infection with the microsporidium (0–30%). This antagonistic effect was driven by a Nosema isolate (termed Nosema sp. Phop) that was purified from microsporidian-infected P. operculella individuals. The purified microsporidium was characterised by morphological features, including size, filament coils and different developmental stages using transmission electron microscopy (TEM). On the molecular level, the partial cistron rDNA information of the small ribosomal subunit (SSU), internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) were identified. Phylogenetic analyses revealed that the newly described microsporidium belongs to the “true Nosema” clade. Partial sequence information of the RNA polymerase II largest subunit (RPB1) suggested that Nosema bombycis is the closest relative (98% identity). The morphological and phylogenetic characteristics suggest that it is an isolate of N. bombycis. Interactions of microsporidia and betabaculoviruses are rarely described in the literature, although mixed infections of different pathogens seem to be rather common events, ranging from antagonistic to mutualistic interactions. The observed antagonistic relationship between the Nosema sp. and PhopGV-GR1.1 showed that pathogen interactions need to be considered when single pathogens are applied to insect populations in the context of biological control of insect pests.

DNA polymerase (DNApol) is highly conserved in all baculoviruses and plays an essential role in viral DNA replication. Previous results showed that the DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) can localize in the nucleus. However, it is not clear how the DNApol is transported into the nucleus. Bioinformatic and GFP localization analysis showed that PiraGV DNApol contains a nuclear localization signal (NLS) at aa 4-25 (LFKRKLDEPPTDHTLVKAIKLS) of the N-terminus that does not match either the classical monopartite or the bipartite NLS consensus sequence. Multiple-point-substitution analysis confirmed that the NLS is required for transport of PiraGV DNApol into the nucleus. We also substituted the NLS of the PiraGV DNApol for that of the alphabaculovirus Spodoptera litura nuclear polyhedrosis virus (SpltNPV) DNApol. A viral growth curve and quantitative real-time PCR revealed that the substitution impaired viral DNA replication and resulted in a reduction in virus production. Together, our results show that PiraGV contains a novel NLS and that the NLS cannot efficiently replace that of SpltNPV DNApol for viral DNA synthesis and virus production.

Baculoviruses have been used as biopesticides for decades. Recently, due to the excessive use of chemical pesticides there is a need for finding new agents that may be useful in biological protection. Sometimes few isolates or species are discovered in one host. In the past few years, many new baculovirus species have been isolated from environmental samples, thoroughly characterized and thanks to next generation sequencing methods their genomes are being deposited in the GenBank database. Next generation sequencing (NGS) methodology is the most certain way of detection, but it has many disadvantages. During our studies, we have developed a method based on Polymerase chain reaction (PCR) followed by Multitemperature Single Stranded Conformational Polymorphism (MSSCP) which allows for distinguishing new granulovirus isolates in only a few hours and at low-cost. On the basis of phylogenetic analysis of betabaculoviruses, representative species have been chosen. The alignment of highly conserved genes—granulin and late expression factor-9, was performed and the degenerate primers were designed to amplify the most variable, short DNA fragments flanked with the most conserved sequences. Afterwards, products of PCR reaction were analysed by MSSCP technique. In our opinion, the proposed method may be used for screening of new isolates derived from environmental samples.

Both Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV) belong to a cluster of four baculoviruses that are infective for different Agrotis species. Belonging further to different baculovirus genera, namely Alphabaculovirus and Betabaculovirus, respectively, AgseNPV-B and AgseGV are candidates to investigate virus interactions in co-infections. However, for the investigation of virus interactions on a cellular level, permissive insect cell-lines are needed. The cell line AiE1611T deriving from Agrotisipsilon eggs has been shown to be permissive for several Alphabaculovirus isolates. In this study, virus replication was followed based on microscopic analysis of infected and transfected cells, as well as on a molecular level by PCR of DNA and cDNA of selected baculovirus transcripts. While the permissivity was not verified for AgseGV, AgseNPV-B produced occlusion bodies in both infection with hemolymph of infected larvae and Lipofectamin transfection with AgseNPV-B genomic DNA. In addition to the possibility to investigate virus interaction of AgseNPV-B with other alphabaculoviruses, the permissivity of AiE1611T for AgseNPV-B further offers the possibility a biological selection to separate AgseNPV-B from AgseGV.

DNA polymerase (DNApol) is present in all baculoviruses and plays a crucial role in viral DNA replication. Previously we showed that the DNApol of the alphabaculovirus group II Spodoptera litura nucleopolyhedrovirus (SpltNPV) could partially substitute for the DNApol of a group I alphabaculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV). However, it is not known if a betabaculovirus DNApol could subsititute for the alphabaculovirus DNApol in AcMNPV. In this report, DNApol of the betabaculovirus Pieris rapae granulovirus (PiraGV) was inserted into a dnapol-null AcMNPV bacmid, creating Bac-AcΔpol:PrPol. The repair virus did not spread to neighboring cells; virus growth curve and real-time PCR revealed that the PiraGV dnapol substitution abrogated AcMNPV DNA replication and virus production. Immunofluorescence microscopy showed that PiraGV DNApol could be expressed and localized to the nucleus. Collectively, our results suggested that the alphabaculovirus AcMNPV DNApol could not be replaced by a DNApol from the betabaculovirus, PiraGV.

The complete genome of a Trichoplusia ni granulovirus (TnGV) is described and analyzed. The genome contains 175,360 bp (KU752557), becoming the third largest genome within the genus Betabaculovirus, smaller only than the Xestia c-nigrum GV (XecnGV) (178,733 pb) and the Pseudaletia unipuncta GV (PsunGV) (176,677 pb) genomes. The TnGV genome has a 39.81% C+G content and a total of 180 ORFs were identified, 96 of them in the granulin gene direction and 84 in the opposite direction. A total of 94.38% of the ORFs showed high identity with those of ClanGV, HaGV, and SlGV. Eight homologous regions (hrs) were identified as well as one apoptosis inhibitor (IAP-3). Interestingly, three viral enhancing factors (VEFs) were located in TnGV genome: VEF-1 (orf153), VEF-3 (orf155), and VEF-4 (orf164), additional to another metalloprotease (orf37). Two ORFs were unique to TnGV (orf100 and orf101) and another one was shared by only TnGV and AgseGV (orf2). Eleven of the deduced proteins showed high identity with proteins from nucleopolyhedroviruses, three with proteins from ascoviruses, and one with an entomopoxvirus protein. The largest deduced protein contains 1,213 amino acids (orf43) and the smallest deduced protein contains only 50 amino acids (orf143). Sequence identity and phylogenetic analyses showed that the closest related genomes to TnGV are, to date, those of PsunGV and XecnGV. This genome analysis may contribute to functional research on TnGV, and may form the bases for the utilization of this betabaculovirus as a pest control agent.

A strategy for biological control of the fall armyworm, Spodoptera frugiperda, has included the use of baculoviruses principally the nucleopolyhedrovirus SfMNPV, which have been extensively characterised. In contrast, the granulovirus of S. frugiperda (SfGV) has been poorly studied even though it is able to enhance the infectivity and virulence of NPVs. In this work, a Colombian SfGV isolate (VG008) was characterised in comparison with a reference isolate from Brazil (VG014). The viral morphology was characterised by ovoidal-shaped occlusion bodies (OB) that contained one single internal virion. Median lethal concentrations (LC50) and mean times to death (MTD) were 4.5 × 105 OBs/mL and 29 days for VG008 and 1.6 × 105 OBs/mL and 33 days for VG014. Both isolates reduced their insecticidal activity by 94%, after one hour of direct irradiation with ultraviolet light type B. The most prominent protein had an apparent molecular mass of 27 kDa and corresponded with the Granulin. Genomic comparison among isolates...