Characterization of Baculoviruses from the Martignoni Collection

Characterization of baculoviruses from the Martignoni collection

George F. Rohrmann Dept. of Microbiology Oregon State University Corvallis, OR 97331‐3804 USA

Phone: 541 737 1793 Email: [email protected]

Rohrmann, G.F., Characterization of baculoviruses from the Martignoni collection. J. Invertebr. Pathol., 2013. 114: p. 61‐64.

Accession Numbers: KC798388‐KC798398 Characterization of baculoviruses from the Martignoni collection

George F. Rohrmann Dept. of Microbiology Oregon State University Corvallis, OR 97331‐3804 USA

Phone: 541 737 1793 Email: [email protected]

Citation: Rohrmann, G.F., Characterization of baculoviruses from the Martignoni collection. J. Invertebr. Pathol., 2013. 114: p. 61‐64.

Accession Numbers: KC798388‐KC798398

Abstract

47 samples from the Martignoni baculovirus collection were characterized by PCR amplification of the lef8 gene. This led to the identification of sequences from viruses that either were not present in the database, or had been identified, but not further characterized. These included an NPV and a GV from Pseudoletia (Mythimna) unipuncta, and NPVs from Coloradia pandora, the oak and hemlock looper (probably Lambdina sp.), Peridroma sp., the pine butterfly (probably Neophasia sp.), Hemileuca sp., Orgyia vetusta, and several Choristoneura sp. A phylogenetic tree was constructed relating these viruses to their closest relatives in the database.

1. Introduction The US Forest Service Laboratory in Corvallis, Oregon developed a biological control program directed against forest insects during the 1960s. Dr. Mauro

1 Martignoni and others collected a variety of diseased insects mostly from the Western US (Arif, 2010). Upon Dr. Martignoni’s retirement, his collection of virus and diseased insects were given to the author. With the advent of PCR amplification, sequence databases, and DNA sequence analysis programs, it has become possible to relatively easily evaluate the novelty of uncharacterized viruses. In this report, PCR amplification of a portion of the lef8 gene was used to determine if there were novel viruses present in this collection that would justify sequencing of their complete genomes.

2. Materials and Methods 2.1. Virus samples The samples were supplied by Mauro Martignoni when he retired in 1986 from the USDA Forest Service Laboratory in Corvallis, OR and included upwards of 200 samples of infected insects and partially purified occlusion bodies collected between 1960 and 1980. They were stored at 4° and arbitrarily assigned numbers (Table 1) for use in this study. When multiple samples with identical sequences were identified, only one was further characterized and included in this study.

2.2. Documentation. In addition to the information in Table 1, the following lists samples that were identical or very similar to each other in the collection, or to other viruses in the database and shows the documentation available. 2.2.1. Pseudoletia (Mythimna) sp. GV #2, 3, 8, 9, 166, and 170. #2 was collected on August 10, 1963. 2.2.2 Coloradia pandora (Pandora pine moth) NPV #6, 19, 152, 157. #19 was from Inyo National Forest, CA June 1981. 2.2.3. Pseudoletia (Mythimna) NPV #7 and 28. #7 was dated 12/13/65. 2.2.4. Pine Butterfly #11 from Missoula MT, July, 1972. 2.2.5. Hemerocampa (Orgyia) vetusta #13 (9/3/69). This virus was not characterized because it was similar to the O. pseudotsugata MNPV (Ahrens et al., 1997). 2.2.6 Oak and hemlock looper (#15 [8/62] and #171 [1970]), respectively. 2.2.7. #17 Heliothis NPV . Collected in Chile (2/29/74), South America. This isolate was not characterized because the sequence data was similar to that from Helicoverpa zea SNPV (NP_542661) (Chen et al., 2001). 2.2.8 Malacosoma sp. NPV #18. Tualatin, OR (10/64). 2.2.9. Choristoneura viridis GV #22 from Gearhart Mt., OR 5/15/79. 2.2.10. Choristoneura murinana NPV #28 described in (Rohrmann et al., 1982). 2.2.11. #164. Malocosoma NPV. 5/1/1968 2.2.12. Pseudohazis (Hemileuca) NPV #165 (probably Hemileuca eglanterina – Western sheep moth) Saturniidae. This virus was likely described in (Hughes, 1978). 2.2.13. Peridroma sp #167 and 168. NPV. 11/26/63 and 6/5/80, respectively.

2.3. DNA template production/purification.

2 The samples consisted of either desiccated or decayed insect carcasses in suspension, or purified or partially purified PIB. The latter samples were alkali dissolved by adding 20 ‐ 50 µl of the sample to 450 µl H2O with 50 µl of 1M Na2CO3, 0.5M NaCl and incubating at 42° for 10 min. The samples were then microcentrofuged for 30 min at room temperature to pellet the virions and resuspended in 50 µl TE and PCR amplified (see below). If no PCR product was evident, the virion sample was extracted using a Qiagen DNAeasy Blood and Tissue Kit and the DNA was then used in a PCR reaction as described below. For samples that were composed of diseased insect carcasses, the polyhedron inclusion bodies (PIB) were partially purified by suspending the material in 0.5% SDS in TE (10mM Tris, 1mM EDTA, pH 8.0) and squeezing them through 4 layers of gauze in a funnel into a 15 ml tube. For analysis, samples of this material were then microfuged briefly and resuspended in TE and then alkali dissolved and processed as described above.

2.4. Polymerase chain reaction (PCR) For PCR, two degenerate primers complementary to the baculovirus lef8 gene were used (Jehle et al., 2006). LEF‐8 is a subunit of the viral RNA polymerase found in all baculoviruses. The primers were fused to a M13 reverse, or T7 promoter primer (underlined below) to allow sequencing of the PCR product. The primers and genome location are as follows: prL8‐2 41,373–41,389 M13 reverse CAGGAAACAGCTATGACCAYRTASGGRTCYTCSGC prL8‐1B 42,075–42,088 T7 TAATACGACTCACTATAGGGCAYGGHGARATGAC A sample was usually diluted 1:200 in TE and 1 µl was used in a 20 µl PCR reaction using a BioRad iQ SYBER Green supermix kit with 1 µl of a 1 µM mixture of the two primers. In this protocol, the heat from the PCR reaction denatures the virion proteins and DNA and makes the DNA template accessible to the Taq polymerase. The PCR reaction was a two step procedure using two different annealing temperatures. 95° 5 min,10 cycles of 94°, 1min; 47°, 1 min; 72°, 1 min; and then 37 cycles of 94°, 20 sec; 52°,45 sec; 72°, 45 sec and then ending with 72 degrees for 10 min. After amplification, 5 ul was examined on a 1% agarose gel to determine if a product of the predicted size was produced. If a product was detected, the remainder of the PCR product (15 ul) was purified using a Qiagen QIAquick pcr purification kit and used for DNA sequencing.

2.4. DNA sequencing and analysis. DNA sequencing was done at the Center for Genome Research and Biocomputing at Oregon State University. The instrumentation and technology employed ABI Prism® 3730 Genetic Analyzer, 3730 Data Collection Software v. 3.0, and DNA Sequencing Analysis Software v. 5 and employed BigDye® Terminator v. 3.1 Cycle Sequencing Kit chemistry. Sequence data was analyzed using the MacVector suite of software programs and all comparisons are based on predicted amino acid sequences.

3. Results and Discussion

3 For this investigation, 47 samples were processed and resulted in the production of 32 PCR products of the appropriate size that were sequenced. Some samples appeared to be mixtures. Readable sequence was produced from 26 samples, some of which were identical to one another. When multiple sequences were identical, a single sample was sequenced from the opposite direction. The virus samples that were characterized are listed in Table 1. The likely species name is indicated and the closest related sequence in Genbank is indicated along with the percent identity. These sequences were subjected to clustalw analysis and a phylogenetic tree was produced (Fig. 1). Three categories of related baculovirus sequences were identified and used for further analysis. These included, i) identical sequences from baculovirus genomes that had been completely sequenced, ii) identical sequences from isolates whose genome had not been sequenced, iii) samples for which there were no identical sequences. From this analysis, the following was evident. 3.1. GV Sequences #2 Pseudoletia (Mythimna) sp. GV was more closely related to the Spodoptera frugiperda GV (76%), than to the Pseudoletia (Mythimna) unipuncta GV sequence (66%) present in the database. #22 Choristoneura viridis GV is identical to the C. occidentalis GV sequence. 3.2. NPV sequences, Group I The pine butterfly sequence (#11), was closest to CfdefNPV (96%). The Choristoneura murinana NPV (ChmuNPV) (#26) was identical to Archips rosanus NPV sequence. The closest related sequenced genome was for CfNPV (93%). The Coloradia pandora NPV (CopaNPV) (#19) was very similar to another isolate of CopaNPV (Jehle et al., 2006). However the most closely related sequenced genome was from CfdefNPV (60%). 3.3. NPV sequences, Group II Pseudoletia (Mythimna) NPV (#7) and Peridroma sp. NPV (#167) are members of the same lineage with #7 identical to a Mythimna unipuncta (MyunNPV) sequence and #167 closely related to Peridroma margaritosa NPV and are 84% identical to each other. The closest sequenced viral genome to these two isolates are the Mamestra configurata‐A NPV (Maco‐A‐NPV) and the Mamestra brassicae MNPV (MabrMNPV) at about 68‐70% identity. The Malacosoma sp. NPV sequences (#18 and #164) were closely related to each other (98%) and those from M. americanum and M. californicum NPV. The most closely related sequenced viral genome is from OrleNPV (61%). The oak and hemlock looper NPVs (#15 and #171, respectively) likely were from insects of the genus Lambdina probably L. fiscellaria. Both have identical LEF‐8 sequences and appear to represent a relatively distinct virus most closely related to Orgyia leucostigma NPV (OrleNV) LEF‐8 (62%). This is in contrast to several Lambdina fiscellaria NPVs that showed similarity to group II OpSNPV sequences (Levin et al., 1997) suggesting that there may be at least two different viruses that infect these species. The Hemileuca sp. isolate (#165) was also a member of the Oak/hemlock looper lineage but was distinct from those viruses (53%) and appear closer to OrleNPV (59%).

4 The following were very close or identical to lef8 sequences from viruses that have already been sequenced. Choc NPV = CfMNPV (de Jong et al., 2005), ChviGV = ChocGV (Escasa et al., 2006), Heliothis NPV = Heliocoverpa zea SNPV (accession # NC_003349), and Hemerocampa (Orgyia) vetusta = Orgyia MNPV (Ahrens et al., 1997).

4. Conclusions This report summarizes a survey of the baculoviruses present in the Martignoni collection and identified at least 10 viruses that showed significant differences from completely sequenced baculovirus genomes present in the database. These 10 samples are now in the process of being completely sequenced. That information should definitively place these viruses within the baculovirus phylogenetic tree, provide further understanding of baculovirus diversity, and provide information on the pathogens of a number of important insects.

5. Acknowledgements This work was supported in part with a grant from the Tartar Fund.

Figure Legends

Fig. 1. Phylogenetic tree showing the relationship of the Martignoni isolates with sequences in GenBank. The asterisk indicates that the complete genome has been sequenced. The numbers are from the Martignoni collection and are defined in Table 1. The NeseNPV sequence was added as an outlier. The tree was calculated using Neighbor Joining; Best Tree; tie breaking = Systematic. Distance: Uncorrected ("p"). Gaps distributed proportionally. Bootstrap values are indicated in bold for values less than 95%. The bootstrap values were calculated using Neighbor Joining; Bootstrap (1000 reps); tie breaking = Systematic. Distance: Uncorrected ("p"). Gaps distributed proportionally. The viruses used for comparison and their abbreviations are as follows: Archips rosanus (ArroNPV), Choristoneura fumiferana (CfMNPV), C. fumiferana def (CfdefMNPV), C. occidentalis (ChocGV), Coloradia pandora (CopaNPV), Euproctis digramma (EudiNPV), Malacosoma americanum (MaamNPV), M. californicum (MacaNPV), Mamestra configurata (MacoNPV‐A), Mythimna unipuncta (MyunNPV), Neodiprion sertifer (NeseNPV), Orgyia leucostigma (OrleNPV)Orgyia pseudotusgata (OpMNPV), Peridroma margaritosa (PemaNPV), Pseudoletia (Mythimna) unipuncta (MyunGV), Spodoptera frugiperda (SfGV). The Genbank accession numbers are indicated next to the virus abbreviation.

Ahrens, C. H., et al., 1997. The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome. Virology. 229, 381‐ 399. Arif, B. M., 2010. Society of Invertebrate Pathology Founders' Lecture, 2010. Mauro Emilio Martignoni: a renaissance scientist in the 20th century. J Invertebr Pathol. 107, 173‐8.

5 Chen, X., et al., 2001. The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome. J Gen Virol. 82, 241‐257. de Jong, J. G., et al., 2005. Analysis of the Choristoneura fumiferana nucleopolyhedrovirus genome. J Gen Virol. 86, 929‐43. Escasa, S. R., et al., 2006. Sequence analysis of the Choristoneura occidentalis granulovirus genome. J Gen Virol. 87, 1917‐33. Harrison, R. L., Popham, H. J., 2008. Genomic sequence analysis of a granulovirus isolated from the Old World bollworm, Helicoverpa armigera. Virus Genes. 36, 565‐81. Hughes, K.M., The macromolecular lattices of polyhedra. J. Invertebr. Pathol., 1978. 31, 217‐ 224. Jehle, J. A., et al., 2006. Molecular identification and phylogenetic analysis of baculoviruses from Lepidoptera. Virology. 346, 180‐193. Levin, D. B., et al., 1997. Characterization of Nuclear Polyhedrosis Viruses from Three Subspecies of Lambdina fiscellaria. J. Invertebr. Pathol. 69, 125‐34. Rohrmann, G. F., et al., 1982. Characterization of DNA from three Nuclear polyhedrosis viruses pathogenic for Choristoneura sp. J. Invertebr. Pathol. 40,

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