RU2798221C1 - Siberian silkworm cytoplasmic polyhedrosis virus strain dendrolimus sibiricus tschetw and an insecticidal drug based on it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the production of biological insecticides for forestry and agriculture based on a new strain of cytoplasmic polyhedrosis virus (CPV), pathogenic for Siberian silkworm (SS) larvae, which are a dangerous pest of larch forests. The strain VTsPSSh-01 of the Siberian silkworm cytoplasmic polyhedrosis virus Dendrolimus sibiricus Tschetw, used to obtain an insecticidal preparation and deposited in the state collection of pathogens of viral infections and rickettsiosis of the FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1109. The insecticidal drug against the Siberian silkworm contains in 1 ml of solution: a suspension of the strain VTsPSSh-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw according to claim 1 with a titer of 2–6×10⁹ polyhedra/ml; glycerin 0.05–0.1 ml; potassium sorbate 1.0–2.0 mg; sodium salt of carboxymethyl cellulose 5.0-10.0 mg; ascorbic acid 0.6-0.8 mg; water — the rest up to 1 ml.

EFFECT: invention makes it possible to increase the effectiveness of the fight against Siberian silkworm (SS) larvae, a dangerous pest of larch forests, and a preparation based on the said strain.

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Description

The invention relates to the microbiological industry and biotechnology, namely the production of biological insecticides for forestry and agriculture based on a new strain of cytoplasmic polyhedrosis virus (CPV), pathogenic for the larvae of the Siberian silkworm (SS), which is a dangerous pest of larch, fir and cedar forests.

The Siberian silkworm cytoplasmic polyhedrosis virus (Dendrolimus sibiricus Tschetw cytoplasmic polyhedrosis virus) belongs to the Cypovirus genus of the Reoviridae family.

The first viral drug against the Siberian silkworm appeared in Russia after a group of researchers traveled to Japan, from where matsukemin, a drug based on the cytoplasmic polyhedrosis virus, was brought (Golosova M.A. Insects - pests of the forest. Biological regulation of populations. M .: MGUL, 2004. 189 Golosova M.A. Nasekomye - vrediteli lesa Biologicheskoe regulirovanie populyatsiy (Insects - forest pests. Biological control of populations) Moscow: MGUL (Moscow St. Univ. For.), 2004. 189 p. (in Russian) ].). The Japanese experience has shown that, in principle, it is possible to obtain an effective viral preparation for protection against cocoonworms of the genus Dendrolimus.

Work on the creation of a domestic drug in Russia based on the cytoplasmic polyhedrosis virus has not been undertaken.

China has experience in large-scale production and use of a viral preparation against silkworms of the genus Dendrolimus based on the cytoplasmic polyhedrosis virus D. punctatus (Sun X., Peng H. Resent advances in biological control of pest insect by using viruses in China // Recent development in research and application of viruses in forest health protection, Pushkino, Beijing, 2010, pp. 96-102). This review describes recent advances in the development of wild and genetically modified viruses as insecticides. A new strategy for the use of insect viruses in China is considered. A report has been published on a cypovirus (CPV) produced in China to control Dendrolimus punctatus, an insect pest of pine forests.

A known strain of cytoplasmic polyhedrosis virus Clanis bilineata tsingtauica (China patent No. CN 112961838, IPC C12N 7/00; A01N 63/00, published 06/15/2021), which is intended for obtaining an insecticidal preparation and is characterized by the fact that it is deposited in the Chinese Collection of Cultures, the deposit address is the Chinese Type Culture Collection Center of Wuhan University, Wuhan, China, the deposit date is September 29, 2020, the object name is Clanis bilineata tsingtauica cytoplasmic polyhedrosis virus (CbCPV), and the deposit number is CCCCC NO: V202064. Clanis bilineata tsingtauica, the two-lined velvet hawk moth, is a moth of the Sphingidae family, a major soybean pest in China, often destroying entire fields. Clanis bilineata tsingtauica cytoplasmic polyhedrosis virus can effectively control the harm caused by Clanis bilineata tsingtauica, continuously acts after a single application for two to three years, and is convenient to manufacture. Cytoplasmic polyhedrosis virus usually has a wide host range, i.e. has a strong insecticidal ability not only against the isolated Sphingidae insect host, but also can kill other insects of the Sphingidae family. After 1 year of continuous use for 2-3 years, the site will no longer have pest outbreaks; the carcasses of larvae breeding with the virus strain will not liquefy, avoiding food for other insects or faecal contamination.

However, the known prototype strain does not affect the larvae of Siberian silkworm insects.

The closest analogue (prototype) for the intended purpose is a strain of bacteria Bacillus thuringiensis var. kurstaki to obtain a lepidocide drug active against silkworm caterpillars, including the Siberian silkworm, deposited in the VKPM collection No. B-5351 (USSR author's certificate No. 1784156, IPC A01N 63/00, publ. 12/30/1992)

The technical result is the creation of a strain of cytoplasmic polyhedrosis virus (CPV), more active and effective against Siberian silkworm (SS) larvae, which are a dangerous pest of coniferous forests, and a preparation based on the specified strain.

The specified technical result is achieved by creating a strain of VTsPSSh-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw, used to obtain an insecticidal preparation and deposited in the state collection of pathogens of viral infections and rickettsiosis of the FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1109. Date of registration 16.09.2021

The specified technical result is also achieved by the creation of an insecticidal preparation against the Siberian silkworm, containing in 1 ml of solution: a suspension of the strain VTSPSSH-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw according to claim 1 with a titer of 2-6×10 ⁹ polyhedra/ml; glycerin 0.05-0.1 ml; potassium sorbate 1.0-2.0 mg; sodium salt of carboxymethyl cellulose 5.0-10.0 mg; ascorbic acid 0.6-0.8 mg; water the rest up to 1 ml.

The virus belongs to the type of cytoplasmic polyhedrosis of the Siberian silkworm (Dendrolimus sibiricus cypovirus 1), to the genus of cytoplasmic polyhedrosis (Cypovirus), the Reoviridae family.

Reoviruses are a family of naked viruses with cubic symmetry virions containing double-stranded fragmented RNA as a genome. The virus infects the epithelial cells of the midgut of the caterpillar, where it forms polyhedra (cytoplasmic protein inclusions consisting of a mass of virions). The virus in polyhedra steadfastly withstands external influences, while virions isolated from them are more sensitive. This applies especially to alcohol, chloroform, deoxycholic (bile) acid, but not to ether or alkali (pH 11) or room temperature, which they tolerate relatively well.

The strain VTsPSh-01 was isolated in 2020. Object of isolation and method of isolation: a dead Siberian silkworm larva collected in the Krasnoyarsk Territory; caterpillar homogenate. Place of isolation (place of biosampling): Krasnoyarsk Territory, Ibreisky District, in a pine forest located 18 km southeast of the village of Stepanovka (55.059652°N, 96.050832°E).

Method for obtaining a strain. The strain was identified in the Federal Budgetary Scientific Institution SSC VB "Vector" of Rospotrebnadzor, Novosibirsk Region, r.p. Koltsovo in January 2021. Isolation was carried out by blind double passaging of the sample on larvae (Dendrolimus sibiricus Tsch.) by infection per os and preparation of a homogenate from dead larvae after each passage. Identification was carried out in March 2021 in the "Collection of Microorganisms" department. The whole genome sequence of the strain was obtained using high-throughput sequencing on the Illumina MiSeq platform. The average coverage of 10 segments was 1924-7894. The genome was assembled by mapping to the reference sequence using the BWA algorithm (v. 0.7.15).

Passage history of the strain. The strain passed 2 passages when infecting caterpillars of the Siberian silkworm laboratory line orally.

Characteristics of the strain.

Cultural and morphological features of the strain. Polyhedra sizes (µm): ∅ 0.8-1.5 µm, virion sizes (nm): 90 nucleocapsids, 40 nm. The strain has a high reproductive activity, well stored at plus 4°C. It is possible to cultivate the virus in vivo on the larvae of the gypsy moth Lymantria dispar L.

Method, conditions and composition of media for reproduction. The cytoplasmic polyhedrosis virus strain VCPSSH-01 is propagated by infecting gypsy moth larvae and further keeping them at a temperature of 25°C and a humidity of 60% on an artificial nutrient medium (IPS) for 18 days. IPA composition: soy flour - 15 g, corn flour - 75 g, fodder yeast - 30 g, ascorbic acid - 4.5 g, benzoic acid - 5.0 ml, agar - 6 g, distilled water - 351 ml.

Activity (productivity) of the strain (indicating the cultivation conditions). The output of the virus is 5×10 ⁸ polyhedra per caterpillar when it is cultivated on 4th instar Lymantria dispar L. larvae.

biochemical activity. Sensitive to alcohol, formalin, chloramine, chloroform; insensitive to ether.

Serological properties: 34 polypeptides, M.v. basic: from 18 to 75 MD.

Genetic characteristics of the strain [established nucleotide sequences of the genome of the strain (length and position of the genome, references to the publication of the established nucleotide sequences, number in GeneBank,)]. Fragmented double-stranded RNA is circular covalently closed. Genome length 24731 bp. The share of GC is 43.28%. Restriction sites (number) for HindIII - 6, EcoRI - 9, PstI - 4, BamH1 - 4 were established. 1 AY211091 .1 AY211093.1 AY211094.1 (Dendrolimus punctatus cypovirus 1) - 673 substitutions.

Virulence (pathogenicity). Human pathogenicity group according to the Russian classification: not pathogenic.

Pathogenicity for animals. It is highly pathogenic for the larvae of the Siberian silkworm Dendrolimus sibiricus Tsch and the gypsy moth Lymantria dispar L.

Pathogenicity for plants: not pathogenic.

Method, conditions and composition of media for long-term storage of the strain.

1. Freeze drying and further storage at T=4°C.

2. In dead caterpillars placed in a 50% aqueous solution of glycerol at T=4°C.

Date and result of last culture viability check. May 12-25, 2021 LD ₅₀ =2×10 ⁴ polyhedra/caterpillar. The activity of the cytoplasmic polyhedrosis virus strain VCPSSH-01 was tested by infecting gypsy moth larvae and further keeping them at a temperature of 25°C and a humidity of 60% on IPS for 18 days.

The date of the proposed reseeding is planned for May 2026. The VTSPSSH-01 strain should be reseeded by infecting gypsy moth larvae and further keeping them at a temperature of 25°C and a humidity of 60% on IPS.

The invention is illustrated by the following graphics. In FIG. 1 shows the caterpillars that died from the VTsPSh-01 virus. a) last instar larva D. sibiricus larva; b) middle-aged larvae of D. sibiricus that died in the experiment; c) Dead larvae of M. sexta of last instar that died in the experiment. In FIG. Figure 2 shows micrographs of the VCPSSH-01 virus strain: a) light microscopy; b) light microscopy using Nomarsky contrast; c) transmission electron microscopy. In FIG. Figure 3 shows whole genome sequence alignments between D. sibiricus VCPSV-01 and available complete genomes of other CPSS type 1 viruses. Sequence discrepancies are indicated by darker shading. (b) Maximum likelihood estimation of topological mismatch between trees of segments 2 and 3 encoding RNA-dependent RNA polymerase (RdRp) and structural protein VP3, respectively. In FIG. Figure 4 shows the virulence of the VTsPSh-01 strain for second-stage larvae of D. sibiricus and L. dispar. a) LD ₅₀ ; b) LV ₅₀ - In FIG. Figure 5 shows the dynamics of mortality of D. sibiricus larvae after infection with the VTsPSh-01 strain. In FIG. 6 shows the host range of VTsPSh-01 mapped on the Lepidoptera phylogenetic tree. The original maximum likelihood tree was taken from [Kawahara, AY et al. Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths. Proc. Natl Acad. sci. USA 116, 22657-22663 (2019)] and folded to family/superfamily level. The status of the analyzed species of Lepidoptera is indicated by red (susceptible species) and gray (resistant species) circles on the leaves of the corresponding family. Non-monophyletic superfamilies are indicated by asterisks. The blue bars represent 95% confidence intervals for node age.

Below are examples of the study of the proposed strain VTsPSSh-01 and a comparison of its activity against the larvae of the Siberian silkworm with the closest analogue for the intended purpose.

Example 1 Isolation of the Siberian silkworm cytoplasmic polyhedrosis virus (Dendrolimus sibiricus cypovirus 1).

The virus was isolated from dead larvae of Dendrolimus sibiricus collected in 2020 in spruce-cedar forests located in the foothills of the Eastern Sayan Mountains (55.059652°N, 96.050832°E). Dead larvae did not liquefy, indicating the absence of nuclear polyhedrosis virus NPV (Fig. 1a). The first identification of the virus was made by microscopic analysis of the contents of dead larvae on a histological slide. For subsequent manipulations with the virus, dead larvae were homogenized in distilled water, and then the virus polyhedra were separated from coarse residues by filtration through gauze, followed by centrifugation at 20,000 g for 20 min. The resulting virus samples were then used for electron microscopy, genomic sequencing, and bioassays.

Example 2 Light Microscopy.

An aqueous suspension of polyhedra was examined under a light microscope (Axioscope 40, Carl Zeiss, Germany) using immersion oil at 1000x magnification. To better visualize the shape of the cytoplasmic polyhedra, Nomarsky contrast was used. Light microscopic analysis of dead larvae revealed many polyhedra with a size of 1.0-2.0 μm (Fig. 2 a, b).

Example 3. Electron microscopy.

The isolated cytoplasmic polyhedra were fixed in suspension by adding an equal volume of 8% paraformaldehyde solution. The mixture was incubated at 4°С for 24 h. Then, the polyhedra were washed with Hank's solution, additionally fixed with 1% osmic acid solution, dehydrated in increasing concentrations of ethanol and acetone, and embedded in the Epon-araldite mixture. Semithin and ultrathin sections were obtained on a Reichert-Jung microtome (Austria). Semi-thin sections were stained with azure-II solution, examined under an Axiolmager Z1 light microscope (ZEISS, Germany), and areas of interest were selected for subsequent ultrastructural analysis. Ultrathin sections were stained with uranyl acetate and lead citrate. Electron microscopy was performed using a JEM 1400 electron microscope (Jeol, Japan). The photographs were taken with a Veleta digital camera (SIS, Germany). Image analysis was performed using iTEM software (SIS, Germany).

Transmission electron microscopy confirmed the presence of spherical viral particles (with an average size of 50-60 nm) covered with a crystalline protein that is typical of the CPV genus. The polyhedra had different shapes (Fig. 2c).

Example 4 Whole genome sequencing.

Nucleic acids were extracted from purified viral polyhedra using the ExtractRNA reagent (ZAO Evrogen, Russia) according to the manufacturer's instructions. Two microliters of an aqueous solution of glycogen (20 mg/ml) was used as a coprecipitant. The precipitate was dissolved in 30 μl of deionized water and used as a template in a reverse transcription reaction for cDNA synthesis according to the SISPA protocol (sequence-independent amplification with a single primer) and according to the method described in the article by Djikeng A. et al. [Djikeng A., Halpin R., Kuzmickas R., Depasse J., Feldblyum J., Sengamalay N., Afonso C, Zhang X., Anderson N.G., and Ghedin E. Viral genome sequencing by random priming methods, 2008 BMC Genomics 9:5.] . The synthesized cDNA fragments were purified using ampoule beads (Beckman Coulter). DNA concentration was determined using a Qubit dsDNA HS assay kit on a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Next generation sequencing libraries were obtained from samples using the NEB Next Ultra II FS DNA Library Prep Kit for Illumina. Paired-end sequencing (2×250 bp) was performed on the MiSeq platform (Illumina) using the MiSeq v2 reagent kit (Illumina), resulting in 637-1038-fold genome coverage.

The genome was assembled in MIRA assembler (v.4.9.6) with default parameters. Multi-sequence alignment of CPV genomes was performed using the MAFFT algorithm (v7.407). Maximum likelihood trees were derived using RAxML-NG [Kozlov, A.M., D. Darriba, T. Flouri, B. Morel, and A. Stamatakis. RAxML-NG: A Fast, Scalable and User-Friendly Tool for Maximum Likelihood Phylogenetic Inference, 2019. Bioinformatics (Oxford, England) 35, no. 21: 4453-4455 (2019).] within the GTR (G+I) model. Branch support was evaluated using the bootstrap method (1000 repetitions).

Prediction of recombination signals and assessment of their statistical significance were carried out using the 3SEQ algorithm [Lam, Η. M., Ratmann, O. & Boni, M. F. Improved algorithmic complexity for the 3SEQ recombination detection algorithm, 2018. Mol. Biol. Evol. 35, 247-251 (2018).].

As in other known representatives of the genus Cypovirus, the sequenced double-stranded RNA genome of VCPSV-01 consists of 10 linear segments, each of which contains one ORF. According to RNA phylogenetic analysis, dependent RNA polymerase (Fig. 3), which is the only conserved protein domain among RNA viruses and is therefore used to determine their evolutionary relationships [Shi, M. et al. Redefining the invertebrate RNA virosphere. Nature 540, 539-543 (2016)], there are only minor differences between VCPS-01 and D. punctatus-1 CPV 1 (DpCPV1) and one of the L. dispar VCV-1 isolates (LdCPV1 ). Although the overall genome is very similar to the HCPV-01 genome, the multiple sequence alignment of the five known CPV1 genomes was rather mosaic: for example, the third segment of HCPV-01 was much closer to LdCPV1 than to DpCPV1 (Fig. 3). Together with the corresponding discrepancy between the phylogenies of the individual segments (Fig. 3), this finding indicates that some recombination events (horizontal transmission) have occurred between different strains of cipoviruses. Accordingly, 3SEQ mosaic analysis predicted that the third segment of HCPN-01 is most likely the result of recombination between HCPS-01 and HCNS-1 (p=3e-276).

Phylogenetic analysis of the sequenced genome of VTsPSh-01 showed that it most likely arose as a result of a recombination event between the DpCPV1 and LdCPV1 strains, involving the replacement of the third segment of the DpCPV1 genome with its LdCPV1 homologue. It has recently been reported that evolutionary events such as reassortment and intragenic recombination are widespread among RNA viruses, including cypoviruses, and serve as a mechanism for adapting to changing environmental conditions, including expansion of the host range [Zhang Zh., Li N., Hou Ch. , Gao K., Tang X. & Guo X. Analysis of reassortant and intragenic recombination in Cypovirus, 2020. Virology Journal 17:48 (2020).]. The possibility of such an evolutionary mechanism is additionally confirmed by the possibility of forming co-infections with several CPV strains in one host, which occurs in natural populations [Pavlushin S.V., Ilinsky Yu.Yu., Belousova I.A., Bayborodin S.I., Lunev E.A., Kechin A.A., Khrapov E.A., Filipenko M.L., Toshchakov S.V., Martemyanov V.V. Appearances are deceptive: Three RNA viruses co-infected with the nucleopolyhedrovirus in host Lymantria dispar, 2021. Virus Research, 297 (2021).]. Therefore, the strain we isolated could well have arisen by recombination of strains of different hosts within the same host, and thus it received a selective advantage due to the expansion of the host spectrum.

Example 5. Determination of LD50 and LV50.

We used a population of D. sibiricus collected in spruce-cedar forests located in the foothills of the Eastern Sayan Mountains. For the bioassay, we obtained the next generation (ie F1) of insects in the laboratory. The larvae were reared at 24° C. and 40-60% relative humidity on a 16/8 (day/night) schedule and fed on 2-year-old spruce shoots Abies sibirica Ledeb.

To detect infection, we conducted a serial breeding experiment. In particular, the following concentrations of virus in suspensions were used: 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ and 10 ³ polyhedra per milliliter. For the inoculation of larvae with the virus, the drip feeding method was used [Hughes PR, van Beek N., Wood Η.A. A modified droplet feeding method for 437 rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae, 1986. J. Invertebr. Pathol. 438, 48:187-192 (1986).]. Second instar larvae were individually given 0.5 µl of virus suspension in 10% sucrose aqueous solution. For better visualization, the drinking suspension was colored with food coloring. The larvae of the control group were given a virus-free solution of sucrose. For each dose, we used 30 individuals. Mortality was recorded daily until pupation. Lethal doses required to kill 50% (LD50) of inoculated larvae were determined using the drc software package [Ritz C, Baty F., Streibig JC, Gerhard D. Dose-response analysis using R, 2015. PLoS ONE 10 (12 ):e0146021 (2015).]. Differences in CD50 were considered statistically significant when the confidence limits did not overlap. Mean time to death (LB50) was determined by Kaplan-Meier survival analysis followed by Logrank test with Holm-Sidak correction.

Infection of the primary host (second instar D. sibiricus larvae) showed an LD50 of 68 polyhedra per larva after 12 days (FIG. 4a). It should be noted that even at the lowest dose of VCPSSH-01 (0.5 polyhedra per larva), a 53% larval mortality rate was recorded throughout the larval stage (FIG. 5). Mature polyhedra have also been found in high concentration in the faeces of infected larvae.

The results obtained showed that the genus Cypovirus contains some species that have prospects as agents for pest control due to their high virulence in relation to the main host, high productivity despite the fact that this virus replicates only in the tissues of the midgut [Chen, Y. , Becnel, J.J., Valles, S.M. RNA Viruses Infecting Pest Insects, 2012. In: Vega, F., Kaya, H.K., editors. insect pathology. 2nd edition. Amsterdam: Elsevier, 133-170 (2012).]. An assessment of the cumulative mortality of D. sibiricus larvae (the main host) showed that even at a dose of only 0.5 polyhedron per larva (i.e., only half of the individuals received the virus), the virus could kill half of the treated population. This high level of infectivity may be due to high susceptibility of the main host or/and successful horizontal transmission of the virus between individuals due to the presence of mature polyhedra in host faeces, as we observed in this study and as previously reported [Marzban R., Not Q., Zhang Q., Liu H.H. Histopathology of cotton bollworm midgut infected with Helicoverpa armigera cytoplasmic polyhedrosis virus, 2013. Braz J Microbiol. 44:1231-1236 (2013).]. The virulence of the new strain, DsCPV1, is much higher than that of another closely related strain, CPV1 (obtained from other hosts [Zhou Y., Qin T., Xiao Y., Qin F., Lei C. Genomic and Biological Characterization of a New Cypovirus Isolated from Dendrolimus punctatus, 2014. PLoS ONE 9(11): el 13201 doi:10.1371/journal.pone.0113201 (2014).]), and the virulence of the CPV genus as a whole [Shapiro M., Im D. J., Adams J. R., Vaughn J. L. Comparative Effectiveness of Lymantria Nuclear Polyhedrosis and Cytoplasmic Polyhedrosis Viruses Against the Gypsy Moth (Lepidoptera: Lymantriidae), 1994. Journal of Economic Entomology 87(l):72-75 (1994).].

Example 6. Productivity of VTsPSSh-01 during its development on the main host.

For this analysis, we used larvae collected in the larch forests of the Baikal region, since the stock of the Sayan population of D. sibiricus in this study was limited. The larvae were grown under the same conditions as described above (except for the host plant) and fed with larch shoots Larix sibirica Ledeb.

For infection, fifth-instar larvae were individually soldered with 2 µl of a suspension of the virus in a stained 10% aqueous sucrose solution. The concentration of the virus in the suspension was 2×10 ⁷ polyhedra/ml. The infected and control groups contained 30 individuals each. Polyhedra were isolated from dead larvae by grinding the larval body in a fixed volume of water. The homogenate suspension (without filtration to avoid loss of polyhedra) was then diluted and the polyhedra were counted on a hemocytometer at 400× magnification. Viral yield was defined as the number of polyhedra per larva.

The average yield of VCPSSH-01 at the time of infection of middle-aged larvae was 10 ⁸ polyhedra per larva with a range of 2.5×10 ⁷ to 3.1×10 ⁸ .

Example 7 Determination of the level of host specificity.

The experiment involved larvae of nine Lepidoptera species from six families (excluding the main host). Oral infection was carried out in two ways: drop feeding and inclusion in the diet. Drop feeding consisted of giving the insects a drop of a virus suspension in a colored 10% sucrose solution. If the larvae refused the sweet drop, then the infection was carried out by contamination of the virus in their nutrient medium. In the latter case, the scheme of infection for all species was as follows: 1 ml of the virus suspension was applied with a brush to the food intended for one group of insects. The amount of contaminated food was such that a group of insects could eat it before it dried. After the larvae had eaten the contaminated food or drunk a drop, they were placed in containers according to the optimal population density for the respective species. Virus concentrations in suspensions during infection of different species were the same; however, when using infection by contamination of the culture medium, the concentration was higher than with drip feeding, in order to adjust the dose for the losses observed with this method, when the larvae do not consume all the treated feed.

A qualitative bioassay of other potential Lepidoptera hosts showed that P. brassicae, M. brassicae, H. cunea, L. dispar and M. sexta are susceptible to HCPS-01 infection, while O. nubilalis, G. mellonella, L. sticticalis, and H. armigera are not susceptible to viral infection.

In this way, HCPS-01 successfully infected six out of 10 Lepidoptera species analyzed, belonging to five families (Erebidae, Sphingidae, Pieridae, Noctuidae and Lasiocampidae), initiating normal pathogenesis in a susceptible host. We present actual sensitive hosts on the Lepidoptera evolutionary tree (Fig. 6), and most of them belong to different families within the Obtectomera clade, a relatively young clade of the order Lepidoptera [Kawahara, A. Y. et al. Phylogenomics reveals the evolutionary timing and pattern of butterflies and moths. Proc. Natl Acad. sci. USA 116, 22657-22663 (2019).]. Pyraloidea_clade shows some pattern of tolerance to the VCPSh-01 strain, although more studies at increased dosages are needed to definitively confirm this claim. A relatively wide host range has been shown for another group 17 cypovirus, strain UsCPV-17, isolated from the mosquito Uranotaenia sapphirina [A. Shapiro, T. Green, S. Rao, S. White, G. Carner, P.P.C. Mertens, JJ. Becnel Morphological and molecular characterization of a Cypovirus (Reoviridae) from the Mosquito Uranotaenia sapphirina (Diptera: Culici-dae) J. Virol., 79 (2005), pp. 9430-9438]. However, in that study, the host range was limited to one family, while in our study it was limited to one order. Thus, the range of hosts of VCPSSH-01 is much wider than it was previously found for other cypoviruses. The virulence of HCPS-01 in relation to alternative hosts was not as high as in relation to the main host, but still comparable to the virulence of other entomopathogens used in pest control [Sun X. History and current status of development and use of viral insecticides in China, 2015. Viruses, 7:306-319 (2015).]. It is known that representatives of the first type of cypovirus were isolated from different Lepidoptera hosts, such as D. punctatus [Zhao S. L., Liang C. Y., Hong J. J., Peng H. Y. Genomic sequence analyzes of segments 1 to 6 of Dendrolimus punctatus cytoplasmic polyhedrosis virus, 2003. Arch Virol. 148(7):1357-68, doi: 10.1007/s00705-003-0103-z (2003).], Bombyx mori [Hukuhara T., Midorikawa M. Compans R.W., Bishop D.H. (eds) Double-stranded RNA viruses: Pathogenesis of cytoplasmic polyhedrosis in the silkworm (Elsevier, New York, 405-414, 1983).] and L. dispar [Pavlushin S.V., Ilinsky Yu.Yu., Belousova I.A., Bayborodin S.I., Lunev E.A., Kechin A.A., Khrapov E.A., Filipenko M.L., Toshchakov S.V., Martemyanov V.V. Appearances are deceptive: Three RNA viruses co-infected with the nucleopolyhedrovirus in host Lymantria dispar, 2021. Virus Research, 297 (2021).], but prior to our study there was no confirmation of such a wide host range for type 1 cypoviruses or even for the Baculoviridae family .

Example 8 Infection of the main host D. sibiricus with a virus passed through the host M. sexta.

Mid-aged M. sexta larvae were infected with the parent virus by spraying the feed. Feed was added at a concentration of 10 ⁷ polyhedra/ml, 1 ml per 20 larvae. Dead larvae were collected and crushed with the addition of distilled water, then the viral particles were separated from coarse residues by filtration through gauze, followed by centrifugation at 20,000 g for 20 min.

Second instar larvae of D. sibiricus were used to test the pathogenicity of the virus passed through M. sexta to the original host. We used the larvae of the daughter generation hatched in the laboratory from the parent population collected in the spruce-cedar forests located in the foothills of the Eastern Sayan. The larvae were reared at 24° C. and 40-60% relative humidity on a 16/8 (day/night) schedule and fed on 2 year old A. sibirica spruce shoots.

For infection, second-instar larvae were individually fed 10 ⁴ polyhedra per larva. The drip feeding method was used [Hughes PR, van Beek N., Wood Η.A. A modified droplet feeding method for 437 rapid assay of Bacillus thuringiensis and baculoviruses in noctuid larvae, 1986. J. Invertebr. Pathol. 438, 48:187-192 (1986).]. Larvae of the second age were soldered with 0.5 µl of a suspension of the virus in a 10% aqueous solution of sucrose. The larvae of the control group were given a virus-free solution of sucrose. The highest dose was chosen to qualitatively confirm the effectiveness of the virus against the main host. Mortality was recorded daily until the last insect died.

The study showed that this virus retained its pathogenicity in relation to the main host: all infected insects (D. sibiricus) died. We also compared this virulence of the VTsPSh-01 strain with its virulence against another alternative host: L. dispar. And this experiment showed that the susceptibility of L. dispar to VCPSSH-01 was significantly lower than that of the main host (Fig. 4 a, b).

Example 9. Compositions of the drug based on the strain VTsPSSh-01 used against the Siberian silkworm.

Composition 1. The content of the insecticidal preparation against the Siberian silkworm in a solution of 1 ml:

suspension of the strain VTsPSSh-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw with a titer of 2×10 ⁹ polyhedra/ml

glycerol 0.05 ml potassium sorbate 1.0 mg sodium carboxymethyl cellulose 5.0 mg ascorbic acid 0.6 mg water the rest up to 1 ml.

Composition 2. The content of the insecticidal drug against the Siberian silkworm in a solution of 1 ml:

a suspension of the strain VTSPSSH-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw with a titer of 6×10 ⁹ polyhedra per 1 ml;

glycerol 0.1 ml; potassium sorbate 2.0 mg; sodium carboxymethyl cellulose 10.0 mg; ascorbic acid 0.8 mg; water the rest up to 1 ml.

The drug is convenient for use in the field using aerosol sprayers, is environmentally friendly and has a selective effect on the target insect - the Siberian silkworm. Composition 1 is used for prevention or at a low degree of infestation of the forest with the Siberian silkworm. Composition 2 is used when the Siberian silkworm infestation of the forest is high.

Example 10. Comparison of the effectiveness of the drug based on the strain VTsPSSh-01 against the Siberian shemoth with the currently used drug Lepidocid SK.

Our studies have shown that to achieve the effectiveness of (73±13)% of this virus against NS, it is required to spend 1.67×10 ¹³ polyhedra/ha. According to our experimental data, the LD50 of VTsPSh-01 against the Siberian silkworm is 68 polyhedra/gus. The test results presented in the passport strain VTSPSSH-01 indicate that LD50 for NSH is 2×10 ⁴ polyhedra/gus. Thus, a suspension of this virus at the same concentration, according to EA, is 295 times more lethal for SS than for NS. Accordingly, to achieve efficiency (73±13)% against SS, it will be necessary to use 5.67×10 ¹⁰ polyhedrons/ha.

Of the biological preparations for the suppression of the Siberian silkworm, Lepidocid SK is currently used. According to the literature data [Fateev Alexander Andreevich "Protection of the forest from the Siberian silkworm in the Kolpashevsky district":

https://vital.lib.tsu.ru/vital/access/manager/Repository/vital:7261:

SIBERIAN FOREST JOURNAL. 2021. No 5. P.9-25 UDC 630*411:595.787 FACTORS OF BIOLOGICAL REGULATION OF SIBERIAN SILKMOTH POPULATIONS AND THEIR USE IN FOREST PROTECTION Yu. I. Gninenko, Yu. N. Baranchikov:

https://siberian forestry journal.rf/upload/iblock/1a3/1a3fadb530f586fe4a b21 e76fb19f509.pdfl efficiency (73±13)% is achieved at a consumption of 3 l of this drug per 1 ha. According to the presented calculations, the same efficiency can be obtained using 0.028 l of the drug based on VTsPSSh-01. Plus, we will have a prolonged effect of controlling the population of the Siberian silkworm due to the vertical transmission of the virus (from one generation of insects to another).

Example 11 Horizontal spread of viral infection among insects.

Epizootics in populations at the stage of population growth do not spontaneously arise. To control the pest, it is necessary to artificially introduce the pathogen into the population. Previously [Kolosov A.V., Kosogova T.A., Bulychev L.E., Sergeev A.N. Ways of horizontal transmission of baculovirus infection in the gypsy moth (Lymantria dispar L.) // Issues of Virology. - 2010. - No. 5. - S. 43-47.] we have shown the ways of horizontal spread of infection of insect viruses. When feeding together with artificially infected insects, the percentage of death of initially uninfected caterpillars was 65%. These data indicate that when carrying out insecticidal treatments, given the ability of larvae for passive migration, it is not necessary to aim at infecting all insects of a given ecosystem. It is enough to create several foci of infection, which will not only become a source of epizootic, but will also contribute to a general decrease in the viability of the pest population. The use of bacterial preparations does not lead to such a spread of infection, since in this case the main function of containment is performed not by the bacteria themselves, but by their metabolic products.

Thus, the presented examples 1-11 indicate the following:

1. The inventive strain VTSPSSH-01 can be considered as a quasi-universal agent for controlling populations of economically important Lepidoptera pests. This means that a versatile viral insecticide manufacturing scheme can be implemented to create a viral product for the broad pest control market. This will positively affect the price of the insecticide and its profitability.

It is possible to mass-produce this strain in vivo in the most economically suitable cheap host species (the current problem with mass cultivation of entomopathogenic viruses in cell lines [media cost] has not yet been resolved). For example, in our study, M. sexta proved to be the best candidate for this purpose because it has large, fast-growing larvae, does not have diapause, and can be grown on a relatively cheap standard diet. Transmission of VTsPSh-01 through M. sexta (i.e. alternative host) retained pathogenicity in relation to the main host. The effectiveness of the drug based on the proposed strain VTsPSSh-01 as an agent for controlling the population of the Siberian silkworm allows it to successfully compete with currently used other biological drugs.

Considering that a viral infection can spread both horizontally and vertically, the use of insecticides based on it can reduce the number of treatments and reduce the cultivated area, while still effectively controlling the target pests.

The above data on VTsPSS-01 should significantly increase the flexibility of the production of viral insecticides and indicate good prospects for VTsPSS-01 as a quasi-universal biological insecticide against lepidopteran pests, since this virus has a true regulatory effect on the dynamics of pest numbers.

Claims (3)

1. Strain VTSPSSH-01 of the Siberian silkworm cytoplasmic polyhedrosis virus Dendrolimus sibiricus Tschetw, used to obtain an insecticidal preparation and deposited in the state collection of pathogens of viral infections and rickettsiosis of the FBSI SRC VB "Vector" of Rospotrebnadzor under the number V-1109. 2. An insecticidal preparation against the Siberian silkworm, including a suspension of the strain VTSPSSH-01 of the virus of cytoplasmic polyhedrosis of the Siberian silkworm Dendrolimus sibiricus Tschetw according to claim 1 with a titer of 2-6×10 ⁹ polyhedra/ml, glycerin, potassium sorbate, sodium salt of carboxymethyl cellulose, ascorbic acid and water with the following quantitative content of components in a solution per 1 ml: suspension of strain VTsPSSh-01 with a titer of 2-6×10 ⁹ polyhedrons/ml glycerol 0.05-0.1 ml potassium sorbate 1.0-2.0 mg carboxymethylcellulose sodium salt 5.0-10.0 mg ascorbic acid 0.6-0.8 mg water the rest up to 1 ml

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