Genetic diversity of viruses is driven by genomic mutations and selection through its host, resulting in differences in virulence as well as host responses. For baculoviruses, which are naturally occurring pathogens of insects and which are frequently sprayed on hundred thousands to millions of hectares as biocontrol agents of insect pests, the phenomenon of virus-host co-evolution is of particular scientific interest and economic importance because high virulence of baculovirus products is essential and emergence of host resistance needs to be avoided as much as possible. In the present study, the population structure of twenty isolates of the Cydia pomonella granulovirus (CpGV), including twelve isolates from different geographic origins and eight commercial formulations, were studied on the basis of next generation sequencing data and by analyzing the distribution of single nucleotide polymorphisms (SNPs). An entirely consensus sequence free quantitative SNP analysis was applied for the identification of 753 variant SNP sites being specific for single as well as groups of CpGV isolates. Based on the quantitative SNP analysis, homogenous, heterogenous as well as mixed isolates were identified and their proportions of genotypes were deciphered, revealing a high genetic diversity of CpGV isolates from around the world. Based on hierarchical clustering on principal components (HCPC) six distinct isolate/group clusters were identified, representing the proposed main phylogenetic lineages of CpGV but comprising full genome information from virus mixtures. The relative location of different isolates in HCPC reflected the proportion of variable compositions of different genotypes. The established methods provide novel analysis tools to decipher the molecular complexity of genotype mixtures in baculovirus isolates, thus depicting the population structure of baculovirus isolates in a more adequate form than consensus based analyses.

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Natural isolates of baculoviruses (as well as other dsDNA viruses) generally consist of homogenous or heterogenous populations of genotypes. The number and positions of single nucleotide polymorphisms (SNPs) from sequencing data are often used as suitable markers to study their genotypic composition. Identifying and assigning the specificities and frequencies of SNPs from high-throughput genome sequencing data can be very challenging, especially when comparing between several sequenced isolates or samples. In this study, the new tool “bacsnp”, written in R programming langue, was developed as a downstream process, enabling the detection of SNP specificities across several virus isolates. The basis of this analysis is the use of a common, closely related reference to which the sequencing reads of an isolate are mapped. Thereby, the specificities of SNPs are linked and their frequencies can be used to analyze the genetic composition across the sequenced isolate. Here, the downstream process and analysis of detected SNP positions is demonstrated on the example of three baculovirus isolates showing the fast and reliable detection of a mixed sequenced sample.

Cydia pomonella granulovirus (CpGV) is successfully used worldwide as a biocontrol agent of the codling moth (CM) (Cydia pomonella). The occurrence of CM populations with different modes of resistance against commercial CpGV preparations in Europe, as well as the invasiveness of CM in China, threatening major apple production areas there, requires the development of new control options. Utilizing the naturally occurring genetic diversity of CpGV can improve such control strategies. Here, we report the identification of seven new CpGV isolates that were collected from infected CM larvae in northwest China. Resistance testing using a discriminating CpGV concentration and the determination of the median lethal concentration (LC50) were performed to characterize their levels of virulence against susceptible and resistant CM larvae. The isolates were further screened for the presence of the 2 × 12-bp-repeat insertion in CpGV gene pe38 (open reading frame 24 [ORF24]), which was shown to be the target of type I resistance. It was found that three isolates, CpGV-JQ, -KS1, and -ZY2, could break type I resistance, although delayed mortality was observed in the infection process. All isolates followed the pe38 model of breaking type I resistance, except for CpGV-WW, which harbored the genetic factor but failed to overcome type I resistance. However, CpGV-WW was able to overcome type II and type III resistance. The bioassay results and sequencing data of pe38 support previous findings that pe38 is the major target for type I resistance. The new isolates show some distinct virulence characteristics when infection of different CM strains is considered. IMPORTANCE CpGV is a highly virulent pathogen of the codling moth (CM). It is registered and widely applied as a biocontrol agent in nearly all apple-growing countries worldwide. The emergence of CpGV resistance and the increasing lack of chemical control options require improvements to current control strategies. Natural CpGV isolates, as well as resistance-breaking isolates selected in resistant CM strains, have provided resources for improved resistance-breaking CpGV products. Here, we report novel CpGV isolates collected in China, which have new resistance-breaking capacities and may be an important asset for future application in the biological control of codling moths.