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

Abstract

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.

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

“…In the case of insect pathogens, the method based on real-time PCR was developed for sensitive detection and quantitative analysis of larvae infected with one granulovirus: EpapGV [37]. The method proposed in this study allows for the discrimination of different GVs and gives comparable results with the one based on the MSSCP technique described by us previously [33] (Figure 6).…”

“…We have previously developed a method based on the Multi-temperature Single Stranded Conformational Polymorphism (MSSCP) technique, which has advantages similar to the one described in this study [ 33 ]. However, the MSSCP-based method has some drawbacks: It requires dedicated equipment, dealing with polyacrylamide gel, and DNA silver staining.…”

“…Although gran, lef-9 and lef-8 share high levels of similarity between GVs, there are some differences in the nucleotide sequences, and to therefore enable the detection of different species, it is necessary to design degenerate universal primers. The primers used for short fragments of granulin (125 bp) and lef-9 (179 bp) previously published by Krejmer-Rabalska et al [ 33 ] (gran-F–5′TAC ATG GTB ACN GAR GA3′, Tm = 43.4–52.2 °C, gran-R-5′AAY TCY TTN CCG CTC CAG TT3′, Tm = 51.9–59.4 °C; lef-9-F-5′CAR AAC AAR AAY GGR TAY GC3′, Tm = 45.9–56.1 °C, lef-9-R-5′GGR TGN CGH GTG TTC CAY AC3′, Tm = 52.7–64.3 °C) were re-evaluated against a broader group of betabaculovirus species (all 24 available, Figure 3a,b) for use in real-time PCR reactions. Degenerate primers were designed to amplify a short fragment of lef-8 (119 bp)—lef-8-F-5′CC K TA Y AT K TT Y TTY AAC AA3′, Tm = 39–50.5 °C, lef-8-R-5′GAT TGA TT D AT R CTC CA3′, Tm = 39.2–46.4 °C (Figure 3c; IUPAC genetic code: B–C or G or T, N–A or T or C or G; R–A or G, Y–C or T, H–A or C or T, K–G or T, D–A or G or T).…”

“…Fast differentiation of a few (or completely new) species in the environmental sample may be quite complex. In the past, our team has developed a method for the differentiation of nucleopolyhedroviruses [ 32 ] and, more recently, for granuloviruses [ 33 ], both based on the Multi-temperature Single-Stranded Conformational Polymorphism (MSSCP) technique [ 34 ]. However, in the current study, we report a method based on real-time polymerase chain reaction (real-time PCR) that allows for differentiation between betabaculoviruses.…”