Identification, characterization and phylogenic analysis of conserved genes within the odvp-6e/odv-e56 gene region of Choristoneura fumiferana granulovirus

The genes that are located within the odvp-6e/odv-e56 region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing the 11 kb BamHI restriction fragment on the ChfuGV genome. The global GC content that was calculated from the data obtained from this genomic region was 34.96%. The open-reading frames (ORFs), located within the odvp-6e/odv-e56 region, are presented and compared to the equivalent ORFs that are located at the same region in other GVs. This region is composed of 14 ORFs, including three ORFs that are unique to ChfuGV with no obvious homologues in other baculoviruses as well as eleven ORFs with homologues to granuloviral ORFs, such as granulin, CfORF2, pk-1, ie-1, odv-e18, p49, and odvp-6e/odv-e56. In this study, the conceptual products of seven major conserved ORFs (granulin, CfORF2, IE-1, ODV-E18, p49 and ODVP-6E/ODV-E56) were used in order to construct phylogenetic trees. Our results show that granuloviruses can be grouped in 2 distinct groups as follows: Group I; Choristoneura fumiferana granulovirus (ChfuGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), and Adoxophyes orana granulovirus (AoGV). Group II; Xestia c-nigrum granulovirus (XcGV), Plutella xylostella granulovirus (PxGV), and Trichoplusia ni granulovirus (TnGV). The ChfuGV conserved proteins are most closely related to those of CpGV, PhopGV, and AoGV. Comparative studies, performed on gene arrangements within this region of genomes, demonstrated that three GVs from group I maintain similar gene arrangements.     

Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).     

Identification and characterization of a putative baculoviral transcriptional factor IE-1 from Choristoneura fumiferana granulovirus

A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV.     

杨扇舟蛾颗粒体病毒晚期表达因子的分析

【目的】本研究对杨扇舟蛾颗粒体病毒(Clostera anachoreta granulovirus,Clan GV)的晚期表达因子(Late expression factors,LEFs)进行生物信息学分析,并对Clan GV的lefs与其它杆状病毒的lefs进行对比,对鉴定lef基因和研究Clan GV的感染机制具有重要意义。【方法】使用ORF finder在线程序进行开放阅读框(ORFs)分析,BLASTP程序用于蛋白序列的同源分析,DNAStar和DNAClub生物学软件用于对序列进行处理和分析,采用BLAST在公共数据库中对序列进行比对,Clustal W Version 1.8和Gene Doc 2.7用于多序列的比对和分析,用MEGA 6.06的NJ法进行系统发育分析。【结果】Clan GV基因组包括15个lef基因,分别为ie-1、lef-2、39k、lef-11、p35、p47、lef-1、lef-6、lef-5、lef-4、dnapol、lef-3、lef-9、lef-8和lef-10。Clan GV与云杉枞色卷蛾颗粒体病毒(Choristoneura occidentalis granulovirus,Co GV)、黄地老虎颗粒体病毒(Agrotis segetum granulovirus,As GV)、苹果异形小卷蛾颗粒体病毒(Cryptophlebia leucotreta granulovirus,Cl GV)、马铃薯块茎蛾颗粒体病毒(Phthorimaea operculella granulovirus,Po GV)和苹果蠹蛾颗粒体病毒(Cydia pomonella granulovirus,Cp GV)有着较近的亲缘关系。颗粒体病毒中只有Clan GV和分月扇舟蛾颗粒体病毒含有p35。Clan GV的lef-2的转录方向不同于其它12个颗粒体病毒。γ和δ杆状病毒分别只含有7个和3个Clan GV的lef同源基因。Clan GV的lefs跟其它杆状病毒lefs的相似性较低,其中Clan GV与其它颗粒体病毒间的相似性高于Clan GV与核型多角体病毒间的相似性。Clan GV的lef-2、39k、p35、lef-5、dnapol和lef-9在启动子上游拥有TATA box;p47和lef-1在TATA下游20~40 bp处还有一个CACT基序;lef-11、p35、lef-6、lef-5、lef-3、lef-9、lef-8和lef-10这8个基因有晚期启动子基序TAAG;而p35、lef-5、lef-9和lef-10上游既有TATA又有TAAG基序。【结论】结果为研究杆状病毒的基因功能和感染机制提供了数据基础。     

Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicted molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with other baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N- and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the ChfuGV odvp-6e/odv-e56 gene. A slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).     

斜纹夜蛾核多角体病毒（SpltNPV）基因组EcoRI—G片段的序列分析

斜纹夜蛾核多角体病毒 (SpltNPV)基因组EcoRI G片段全长 745 0bp ,包括 7个开放读码框 :vp39、lef 4、cg30、p91、vp33、tlp2 0、AcMNPVORF81同源基因和一个同源区 (hr)。基因组排列比较分析发现 ,这些基因在基因组中的排列 ,在所有已知序列的杆状病毒中都比较保守     

中国棉铃虫单粒包埋核多角体病毒（HaSNPV）HindⅢ-Ⅰ片段的序列分析与基因排列研究

对中国棉铃虫单粒包埋核多角体病毒（HaSNPV)基因组中的HindⅢ-Ⅰ片段的序列进行分析,该片段全长7501nt,包括10个开放阅读框：AcMNPV ORF111的同源基因（Ac 111),晚期表达因子2（lef-2）基因,病毒核壳结构蛋白p24基因、gp16基因、多角体包膜蛋白（polyhedron envelope protein,pep)基因、AcMNPV ORF63的同源基因（Ac63),38.7kD基因,晚期表达因子1（lef-1) 基因,及两个特有基因HaSNPV orf591和HasSNPV orf435。基因组的排列比较分析发现与AcMNPV有较大区别。     

油桐尺蠖单粒包埋核型多角体病毒（BusuNPV）BamHI—H片段的序列分析

对油桐尺蠖单粒包埋核型多用体病毒（Buzurasuppressariasingle－nucleocapsidnucleopolyhedrovirus,BusuNPV）基因组中BamHI－H片段的序列进行分析,该片段全长2422bp,包括三个开放阅读框：p47基因（AcMNPVORF40的同源区）的5′端,完整的组织蛋白酶基因（cathepsin）（AcMNPVORF127的同源区）和p74基因（AcMNPVORF138的同源区）的3′端。序列比较分析表明,BusuNPV的这三个基因与其它杆状病毒的同源基因具有相同的结构保守区。BusuNPV基因组BamHI－H片段上这三个基因的排列顺序完全不同于AcMNPV相应基因的排列顺序。     

Genome sequencing and analysis of a granulovirus isolated from the Asiatic rice leafroller,Cnaphalocrocis medinalis

The complete genome of Cnaphalocrocis medinalis granulovirus(CnmeGV) from a serious migratory rice pest, Cnaphalocrocis medinalis(Lepidoptera: Pyralidae), was sequenced using the Roche 454 Genome Sequencer FLX system(GS FLX) with shotgun strategy and assembled by Roche GS De Novo assembler software. Its circular double-stranded genome is 111,246 bp in size with a high A+T content of 64.8% and codes for 118 putative open reading frames(ORFs). It contains 37 conserved baculovirus core ORFs, 13 unique ORFs, 26 ORFs that were found in all Lepidoptera baculoviruses and 42 common ORFs. The analysis of nucleotide sequence repeats revealed that the CnmeGV genome differs from the rest of sequenced GVs by a 23 kb and a 17 kb gene block inversions, and does not contain any typical homologous region(hr) except for a region of non-hr-like sequence. Chitinase and cathepsin genes, which are reported to have major roles in the liquefaction of the hosts, were not found in the CnmeGV genome, which explains why CnmeGV infected insects do not show the phenotype of typical liquefaction. Phylogenetic analysis,based on the 37 core baculovirus genes, indicates that CnmeGV is closely related to Adoxophyes orana granulovirus. The genome analysis would contribute to the functional research of CnmeGV,and would benefit to the utilization of CnmeGV as pest control reagent for rice production.     

Characterization of a granulovirus from the cassava hornworm (Erinnyis ello: Sphingidae)

A Colombian isolate of Erinnyis ello granulovirus (EeGV) was characterized by electron microscopy, restriction endonuclease digestion, and SDS-PAGE. Electron microscopy showed the occlusion bodies to have a morphology typical of granuloviruses. The restriction patterns of DNA from EeGV and the granuloviruses of Trichoplusia ni (TnGV) and Pieris rapae (PrGV) show little or no similarity, indicating little relatedness among these viruses. EeGV was estimated to possess a relatively small genome of 90.5 +/- 0.5 kbp. SDS-PAGE analysis compared the occulsion body and enveloped nucleocapsid proteins of EeGV and TnGV, and the polypeptide patterns also showed little similarity between these viruses. These analyses, as well as comparison of our results to those reported for other granuloviruses, indicate that EeGV represents a new granulovirus isolate.     

柞蚕核型多角体病毒泛素类似基因的克隆与序列分析

从感病的柞蚕Antheraea pernyi蛹中分离纯化柞蚕核型多角体病毒 (ApNPV),提取基因组DNA,分别构建ApNPV DNA的HindⅢ和SalⅠ酶切片段文库。对基因文库中1个克隆进行序列分析,得到1个长度为321 bp的序列,其中包含一个编码76个氨基酸的开放阅读框,预测的分子量为8.46 kD,系泛素类似基因。在读码框的上游调控序列中,具有典型的晚期基因启动子序列ataag。氨基酸序列同源性分析结果表明,ApNPV与黄杉毒蛾Orgyia pseudotsugata核型多角体病毒 (OpNPV)的同源性最高 (96.1%),与苜蓿尺蠖Autographa californica核型多角体病毒 (AcNPV) 的同源性为86.8%,与棉褐带卷蛾Adoxophyes orana 颗粒体病毒 (AoGV) 的同源性最低（71.1%）,但与人类、线虫和酵母的泛素同源性分别为77.6%、76.3%和76.3%。一些氨基酸残基在真核生物中保守,在杆状病毒中不保守,个别氨基酸残基是杆状病毒所特有的,这些氨基酸序列的改变对杆状病毒泛素基因的作用有待进一步研究。     

Sequence and organization of the Neodiprion lecontei nucleopolyhedrovirus genome

All fully sequenced baculovirus genomes, with the exception of the dipteran Culex nigripalpus nucleopolyhedrovirus (CuniNPV), have previously been from Lepidoptera. This study reports the sequencing and characterization of a hymenopteran baculovirus, Neodiprion lecontei nucleopolyhedrovirus (NeleNPV), from the redheaded pine sawfly. NeleNPV has the smallest genome so far published (81,755 bp) and has a GC content of only 33.3%. It contains 89 potential open reading frames, 43 with baculovirus homologues, 6 identified by conserved domains, and 1 with homology to a densovirus structural protein. Average amino acid identity of homologues ranged from 19.7% with CuniNPV to 24.9% with Spodoptera exigua nucleopolyhedrovirus. The conserved set of baculovirus genes has dropped to 29, since NeleNPV lacks an F protein homologue (ac23/ld130). NeleNPV contains 12 conserved lepidopteran baculovirus genes, including that for DNA binding protein, late expression factor 11 (lef-11), polyhedrin, occlusion derived virus envelope protein-18 (odv-e18), p40, and p45, but lacks 21 others, including lef-3, me53, immediate early gene-1, lef-6, pp31, odv-e66, few polyhedra 25k, odv-e25, protein kinase-1, fibroblast growth factor, and ubiquitin. The lack of identified baculovirus homologues may be due to difficulties in identification, differences in host-virus interactions, or other genes performing similar functions. Gene parity plots showed limited colinearity of NeleNPV with other baculoviruses, and phylogenetic analysis indicates that NeleNPV may have existed before the lepidopteran nucleopolyhedrovirus and granulovirus divergence. The creation of two new Baculoviridae genera to fit hymenopteran and dipteran baculoviruses may be necessary.     

Genome sequence analysis and organization of the Hyphantria cunea granulovirus (HycuGV-Hc1) from Turkey

The fall webworm (Hyphantria cunea) impacts a wide variety of crops and cultivated broadleaf plant species. The pest is native to North America, was introduced to Europe and has since spread further as far as central Asia. Despite several attempts to control its distribution, the pest continues to spread causing damage all over the world. A naturally occurring baculovirus, Hyphantria cunea granulovirus (HycuGV-Hc1), isolated from the larvae of H. cunea in Turkey appears to have a potential as microbial control agent against this pest. In this report we describe the complete genome sequence and organization of the granulovirus isolate (HycuGV-Hc1) that infects the larval stages and compare it to other baculovirus genomes. The HycuGV-Hc1 genome is a circular double-stranded DNA of 114,825 bp in size with a nucleotide distribution of 39.3% G + C. Bioinformatics analysis predicted 132 putative open reading frames of (ORFs) ≥ 150 nucleotides. There are 24 ORFs with unknown function. Seven homologous repeated regions (hrs) and two bro genes (bro-1 and bro-2) were identified in the genome. Comparison to other baculovirus genomes, HycuGV-Hc1 revealed some differences in gene content and organization. Gene parity plots and phylogenetics confirmed that HycuGV-Hc1 is a Betabaculovirus and is closely related to Plutella xylostella granulovirus. This study expands our knowledge on the genetic variation of HycuGV isolates and provides further novel knowledge on the nature of granuloviruses.     

Genomic sequence analysis of granulovirus isolated from the tobacco cutworm, Spodoptera litura

Background

Spodoptera litura is a noctuid moth that is considered an agricultural pest. The larvae feed on a wide range of plants and have been recorded on plants from 40 plant families (mostly dicotyledons). It is a major pest of many crops. To better understand Spodoptera litura granulovirus (SpliGV), the nucleotide sequence of the SpliGV DNA genome was determined and analyzed.

Methodology/Principal Findings

The genome of the SpliGV was completely sequenced. The nucleotide sequence of the SpliGV genome was 124,121 bp long with 61.2% A+T content and contained 133 putative open reading frames (ORFs) of 150 or more nucleotides. The 133 putative ORFs covered 86.3% of the genome. Among these, 31 ORFs were conserved in most completely sequenced baculovirus genomes, 38 were granulovirus (GV)-specific, and 64 were present in some nucleopolyhedroviruses (NPVs) and/or GVs. We proved that 9 of the ORFs were SpliGV specific.

Conclusions/Significance

The genome of SpliGV is 124,121 bp in size. One hundred thirty-three ORFs that putatively encode proteins of 50 or more amino acid residues with minimal overlap were determined. No chitinase or cathepsin genes, which are involved in the liquefaction of the infected host, were found in the SpliGV genome, explaining why SpliGV-infected insects do not degrade in a typical manner. The DNA photolyase gene was first found in the genus Granulovirus. When phylogenic relationships were analyzed, the SpliGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XecnGV), which belong to the Type I-granuloviruses (Type I-GV).     

Horizontal Escape of the Novel Tc1-Like Lepidopteran Transposon TCp3.2 into Cydia pomonella Granulovirus

We characterized an insertion mutant of the baculovirus Cydia pomonella granulovirus (CpGV), which contained a transposable element of 3.2 kb. This transposon, termed TCp3.2, has unusually long inverted terminal repeats (ITRs) of 756 bp and encodes a defective gene for a putative transposase. Amino acid sequence comparison of the defective transposase gene revealed a distant relationship to a putative transposon in Caenorhabditis elegans which also shares some similarity of the ITRs. Maximum parsimony analysis of the predicted amino acid sequences of Tc1- and mariner-like transposases available from the GenBank data base grouped TCp3.2 within the superfamily of Tc1-like transposons. DNA hybridization indicated that TCp3.2 originated from the genome of Cydia pomonella, which is the natural host of CpGV, and is present in less than 10 copies in the C. pomonella genome. The transposon TCp3.2 most likely was inserted into the viral genome during infection of host larvae. TCp3.2 and the recently characterized Tc1-like transposon TC14.7 (Jehle et al. 1995), which was also found in a CpGV mutant, represent a new family of transposons found in baculovirus genomes. The occasional horizontal escape of different types of host transposons into baculovirus genomes evokes the question about the possible role of baculoviruses as an interspecies vector in the horizontal transmission of insect transposons. Received: 27 February 1997 / Accepted: 16 May 1997