Elucidating the genetic diversity of Phthorimaea operculella granulovirus (PhopGV).

Twelve complete genome sequences of Phthorimaea operculella granulovirus (PhopGV) isolates from four different continents (Africa, South America, Asia and Europe) were analysed after Illumina next-generation sequencing (NGS). The isolates have a circular double-stranded DNA genome that is 118 355 to 119 177 bp in length and all of them encode 130 open reading frames (ORFs). Analysis of single-nucleotide polymorphisms (SNPs) revealed a unique set of SNP positions for every tested isolate. The genome sequences of the investigated PhopGV isolates were classified into a new system of four (1-4) groups according to the presence of group-specific SNPs as well as insertions and deletions. These genome groups correlated with phylogenetic lineages inferred from minimum-evolution trees of the whole-genome consensus nucleotide sequences. All members of group 3 originated from the Mediterranean area, whereas the geographical origin and the group assignment did not correlate for isolates belonging to genome groups 1, 2 or 4. The high degree of coverage facilitated the determination of variant nucleotide frequencies. We conclude that the geographical isolates of PhopGV are genetically highly similar. On the other hand, they were rarely genetically homogenous and in most cases appeared to be mixtures of multiple genotypes.

Interaction of Phthorimaea operculella granulovirus with a Nosema sp. microsporidium in larvae of Phthorimaea operculella.

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.

Genome-wide diversity in temporal and regional populations of the betabaculovirus Erinnyis ello granulovirus (ErelGV).

BACKGROUND: Erinnyis ello granulovirus (ErelGV) is a betabaculovirus infecting caterpillars of the sphingid moth E. 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. RESULTS: No 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 of p10, fgf, and pep. Another important contrast on sequence diversity was found on genes encoding complex subunits and/or involved in the same biological processes, as late expression factors (lefs) and per os infectivity factors (pifs). Interestingly, several polymorphisms in coding regions lie on sequences encoding specific protein domains. CONCLUSIONS: By 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.

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide.

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebialeucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as "stable", "reversion", and "unstable switching". Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Multitemperature Single Stranded Conformational Polymorphism.

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.

Bacmid Expression of Granulovirus Enhancin En3 Accumulates in Cell Soluble Fraction to Potentiate Nucleopolyhedrovirus Infection.

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.

Gene expression patterns of Cydia pomonella granulovirus in codling moth larvae revealed by RNAseq analysis.

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.

Single nucleotide polymorphism (SNP) frequencies and distribution reveal complex genetic composition of seven novel natural isolates of Cydia pomonella granulovirus.

The co-evolution between baculoviruses and their insect hosts results in selection of virus populations. To explore this phenomenon at the molecular level, seven natural isolates of Cydia pomonella granulovirus (CpGV) collected from orchards in northwest China were studied using Illumina next generation sequencing (NGS). A total of 540 genome positions with single nucleotide polymorphisms (SNPs) were detected in comparison with known CpGV isolates. New members of previously defined phylogenetic genome groups A, D and E of CpGV, as well as two novel phylogenetic lines, termed genome group F and G, were identified. Combining SNP frequency distribution with the prevalence of genome group-specific SNPs, revealed that six isolates of CpGV were mixtures of different ratios of at least two genotypes, whereas only one isolate, CpGV-WW, was genetically highly homogeneous. This study significantly extends our current understanding of the genetic diversity of CpGV and opens new lines of application of this virus.

New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR).

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.

Partial Loss of Inheritable Type I Resistance of Codling Moth to Cydia pomonella qranulovirus.

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.