Sequence analysis of the Xestia c-nigrum granulovirus genome.

The nucleotide sequence of the Xestia c-nigrum granulovirus (XcGV) genome was determined and found to comprise 178,733 bases with a G+C content of 40.7%. It contained 181 putative genes of 150 nucleotides or greater that showed minimal overlap. Eighty-four of these putative genes, which collectively accounted for 43% of the genome, are homologs of genes previously identified in the Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) genome. These homologs showed on average 33% amino acid sequence identity to those from AcMNPV. Several genes reported to have major roles in AcMNPV biology including ie-2, gp64, and egt were not found in the XcGV genome. However, open reading frames with homology to DNA ligase, two DNA helicases (one similar to a yeast mitochondrial helicase and the other to a putative AcMNPV helicase), and four enhancins (virus enhancing factors) were found. In addition, several ORFs are repeated; there are 7 genes related to AcMNPV orf2, 4 genes related to AcMNPV orf145/150, and a number of repeated genes unique to XcGV. Eight major repeated sequences (XcGV hrs) that are similar to sequences found in the Trichoplusia ni GV genome (TnGV) were found.     

Homologous region 1 of Plutella xylostella granulovirus functions as an enhancer for early gene expression

Homologous regions (hrs) contained in genomes of baculoviruses have been shown to function as origins of viral DNA replication in alphabaculoviruses and betabaculoviruses, and as enhancers for early gene expression in alphabaculoviruses. The hr sequences of betabaculoviruses differ substantially from the ones of alphabaculoviruses. The enhancing property of betabaculovirus hrs has not been reported. In this study, transient assays were performed to investigate the effects of Plutella xylostella granulovirus (PlxyGV) hr1 on early gene expression of the virus. It was shown that hr1 stimulated reporter gene expression from the promoters of four early genes – ie1, dnapol, lef1, and lef9 – independent of additional viral gene expression. The PlxyGV ie1 was shown to repress reporter gene expression from all four early gene promoters in a Trichoplusia ni cell line, both in the presence and absence of hr.     

Characterization and partial genome sequence analysis of Clostera anachoreta granulovirus

The morphological and biological properties as well as partial genomic sequencing of a granulovirus isolated from Clostera anachoreta (Lepidoptera: Notodontidae), C. anachoreta granulovirus (ClanGV), were carried out. The ovoidal occlusion bodies were 337 nm x 170 nm in size, and each granule contained one single rod-shape virion, with a mean size of 250 nm x 46 nm. Granulin had a molecular weight of approximately 30 kDa. ClanGV genome size was estimated as 104.34 kb based on the restriction fragments. The restriction pattern of the ClanGV genome was different from other GVs. A restriction fragment genomic library of ClanGV genome was constructed. The library consisted of nine SalI fragments, seven HindIII fragments and seven EcoRI fragments. One 4.8 kb fragment of the genome, digested by SalI, was sequenced and analyzed. This region was composed of eight unknown ORFs, two baculoviruses homologous gene (vp1054 and lef10) and partial sequence of lef-8. The unknown ORFs included three unique to ClanGV, the other five ORFs were related to baculoviruses. The ORFs, located within this restriction fragment, were compared to homologues in other GVs. The results indicated that ClanGV, CpGV, ClGV, AoGV and PoGV had similar arrangement and orientation of the homologous ORFs. Phylogenetic analysis of VP1054 proteins from 20 baculoviruses indicated that ClanGV was more closely related to CpGV, ClGV, AoGV and PoGV than to other baculoviruses.     

The genome of the Cryptophlebia leucotreta granulovirus

The genome of the Cryptophlebia leucotreta granulovirus (CrleGV) was sequenced and analyzed. The double-stranded circular genome contains 110907 bp and potentially encodes 129 predicted open reading frames (ORFs), 124 of which were similar to other baculovirus ORFs. Five ORFs were CrleGV specific and 26 ORFs were common to other granulovirus genomes. One ORF showed a significant similarity to a nonstructural protein of Bombyx mori densovirus-2. A baculovirus chitinase gene was identified, which is most likely not functional, because its central coding region including the conserved chitinase active site signature is deleted. Three gene copies (Crle20, 23, and 24) containing the Baculo PEP N domain of the polyhedron envelope protein were identified in CrleGV and other GV genomes. One of them (Crle23) appeared also to contain a p10-like sequence encoding of a number of leucine-rich heptad repeats and a proline-rich domain. Another striking feature of the genome is the presence of a hypervariable non-hr ori-like region of about 1800 bp consisting of different kinds of repeats and palindromes. Three other repeat-rich regions were identified within the genome and are considered as homologous regions (hrs). CrleGV is most closely related to the Cydia pomonella granulovirus (CpGV) as revealed by genome order comparisons and phylogenetic analyses. However, the AT content of the CrleGV genome, which is 67.6% and the highest found so far in baculoviruses, differed by 12.8% from the AT content of CpGV. This resulted in a major difference in the codon usage of both viruses and may reflect adaptive selection constraints to their particular hosts.     

The complete sequence of the Adoxophyes orana granulovirus genome

The nucleotide sequence of the Adoxophyes orana granulovirus (AdorGV) DNA genome was determined and analysed. The genome contains 99,657 bp and has an A + T content of 65.5%. The analysis predicted 119 ORFs of 150 nucleotides or larger that showed minimal overlap. Of these putative genes, 104 (87%) were homologous to genes identified previously in other baculoviruses. The mean overall amino acid identity of AdorGV ORFs was highest with CpGV ORFs at 48%. Sixty-three ORFs were conserved among all lepidopteran baculoviruses and are considered to be common baculoviral genes. Several genes reported to have major roles in baculovirus biology were not found in the AdorGV genome. These included chitinase and cathepsin, which are involved in the liquefaction of the host, which explains why AdorGV-infected insects do not degrade in a typical manner. The AdorGV genome encoded two inhibitor of apoptosis (iap) genes iap-3 and iap-5. Among all of the granuloviruses genomes there was a very high level of gene collinearity. The genes shared by AdorGV and CpGV had exactly the same order along the genome with the exception of one gene, iap-3. The AdorGV genome did not contain typical homologous region (hr) sequences. However, it contained nine repetitive regions in the genome.     

The p35 and ie1 of Autographa californica multiple nucleopolyhedrovirus could rescue late gene expression of Plutella xylostella granulovirus in nonpermissive cell lines

There are no stable permissive cell lines available for in vitro replication of PlxyGV. In this study, several PlxyGV bacmids containing egfp and plasmids expressing the luciferase gene (luc) were constructed and used to transfect insect cell lines from Plutella xylostella, Trichoplusia ni (Hi5), and Spodoptera frugiperda. Fluorescence was observed only in the cells transfected with a bacmid with egfp driven by a PlxyGV ie1 promoter, but not by a PlxyGV vp39 or granulin promoter. In transient assays, various levels of LUC activity were detected in the cells transfected with individual reporter plasmids containing the luc driven by the promoters of PlxyGV early genes ie1, exon0, dnapol, lef1, lef9, and orf105, suggesting that the PlxyGV early genes could be activated in the cells independent of virus infection. The addition of a PlxyGV bacmid in the transfections activated luc expression from the promoters of PlxyGV late genes vp39 and granulin only at minimum levels, and caused significant reduction in luc expression from the early promoters, may be due to apoptosis triggered by the PlxyGV bacmid. PlxyGV reporter bacmids containing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes p35 or p35 and ie1 or p35, ie1 and gp64 expressed LUC from a PlxyGV vp39 promoter at levels of 2.6, 8.3, and 23 times higher than those produced by the basic PlxyGV reporter bacmid, respectively, in transfected Hi5 cells. Green fluorescence was present in the cultures of all three cell lines transfected by a PlxyGV bacmid containing egfp with a vp39 promoter and AcMNPV ie1, p35, and gp64 with their native promoters. The fluorescence was also observed in the culture of Hi5 cells inoculated with the supernatant from the transfection. These results suggest that AcMNPV p35 could rescue late gene expression, and the ie1, p35, and gp64 may cooperatively rescue replication of PlxyGV in the cells.     

Complete genome sequence of Agrotis segetum granulovirus Shanghai strain

The complete nucleotide sequence of Agrotis segetum granulovirus Shanghai strain (AgseGV-L1) was determined and compared with that of AgseGV Xinjiang strain (AgseGV-XJ). The circular genome of AgseGV-L1 is 131,442 bp and has a G+C content of 37.27 %. It includes 149 ORFs, 24 of which are unique to AgseGV (AgseGV-L1 and AgseGV-XJ [GenBank accession no. NC_005839]). The average level of amino acid sequence identity between AgseGV-L1 and other granulovirus (GV) homologues (except AgseGV-XJ) ranged from 44.3 % (Adoxophyes orana granulovirus [AdorGV]) to 49 % (Cydia pomonella granulovirus [CpGV]). The AgseGV-L1 genome is 99 % identical to that of AgseGV-XJ. They have 196 differences, including 172 substitutions, 21 deletions and 3 insertions. Two homologous regions (hrs) were detected in two intergenic spaces, which share low identity and both lack a palindromic core. A p6.9 gene was found in this genome, which shared 38 %-59 % amino acid identity with those of other baculoviruses. No differences were found in the hr and p6.9 sequences of AgseGV-L1 and AgseGV-XJ. Ie-1 is a known immediate-early gene, but AgseGV-L1 ie-1 has no recognizable promoter element. By BLASTP analysis, one bro gene homologue of NPVs was detected (Agse148). Phylogenetic analysis based on the 29 core baculovirus genes indicated that AgseGV-L1 is closely related to AgseGV-XJ, Helicoverpa armigera granulovirus (HearGV), Helicoverpa armigera granulovirus (XecnGV), Pseudaletia unipuncta granulovirus (PsunGV), Spodoptera litura granulovirus (SpliGV) and Plutella xylostella granulovirus (PlxyGV).     

Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G+C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2-29 palindromic repeats, with an average length of 534 bp and base content (G+C%) of 33.0.     

Sequence analysis of the 3'-end of feline calicivirus genome.

The nucleotide sequence of the 3'-end of the Japanese F4 strain of feline calicivirus (FCV) RNA was determined from a cloned cDNA of 3.5 kbp. We found three open reading frames (ORFs). The largest ORF encoded a 668-amino acid protein of 73,588 Da, which was presumably the capsid precursor protein of FCV and had significant amino acid sequence homology with the VP3 of picornaviruses. A small ORF at the extreme 3'-end was compared with that of the F9 strain of FCV, a vaccine strain originally from the U.S. Highly conserved amino acid sequences were shown, suggesting that this ORF might be functional and encode a putative 106-amino acid protein of 12,153 Da. The other ORF in the 5'-flanking region of the cDNA had consensus amino acid sequences conserved among the RNA-dependent RNA polymerases.     

Analysis of the immune-inducible genes of Plutella xylostella using expressed sequence tags and cDNA microarray

In the present study, the complex gene expression responses of Plutella xylostella to microbial challenges and injury were surveyed using a newly constructed expressed sequence tag (EST) clone collection and cDNA microarray analysis. A total of 1132 P. xylostella ESTs were cloned, annotated and categorized by their putative functions; these included proteases, protease inhibitors, recognition molecules and anti-microbial peptides. GeneOntology revealed that 4% of the P. xylostella ESTs corresponded to immunity-related genes potentially involved in innate immunity. We then used microarray analysis to identify 44 genes that were differentially expressed with at least a two-fold expression difference in P. xylostella before and after pathogen challenge. Together, our EST categorization and microarray profiling analyses allowed us to identify 70 genes that should be considered candidate immune response genes, providing important new insights into the molecular events that occur during the innate immune response in P. xylostella.     

Sequence and phylogenetic analysis of the S-class genome segments of a duck orthoreovirus

In spite of common properties duck orthoreoviruses (DRVs) are antigenically different from other avian orthoreoviruses (ARVs). We analyzed the S-class genome segments of the DRV S12 and compared them with S-class genome segments of other orthoreoviruses compared to those of ARV S1133. The full-length S-class sigmaA, sigmaB, sigmaNS, and sigmaC genes were determined and compared with other ARVs to study the degree of genetic divergence and evolution of DRVs. The alignment of the DRV S12 sigmaA, sigmaB, sigmaNS, and sigmaC genes with DRV 89026 showed 90.0%, 93.6%, 88.0%, and 93.1% nucleotide identity and 97.1%, 94.3%, 95.8%, and 93.7% amino acid identity, respectively. The alignment of the DRV S12 sigmaA, sigmaB, sigmaNS, and sigmaC genes with other ARVs revealed 76.0-77.1%, 52.5-55.1%, 78.4-79.6%, and 2.7-9.9% nucleotide identity and 89.5-91.2%, 61.4-62.0%, 91.6-92.7% and 22.6-26.7% amino acid identity, respectively. Phylogenetic analyses demonstrated that DRVs were quite different from other ARVs and provided the evidence for the diversity among avian orthoreoviruses.     

Sequence and phylogenetic analysis of the S1 genome segment of turkey-origin reoviruses

Based on previous reports characterizing the turkey-origin avian reovirus (TRV) σB (σ2) major outer capsid protein gene, the TRVs may represent a new group within the fusogenic orthoreoviruses. However, no sequence data from other TRV genes or genome segments has been reported. The σC protein encoded by the avian reovirus S1 genome segment is the cell attachment protein and a major antigenic determinant for avian reovirus. The chicken reovirus S1 genome segment is well characterized and is well conserved in viruses from that species. This report details the amplification, cloning and sequencing of the entire S1 genome segment from two and the entire coding sequences of the σC, p10 and p17 genes from an additional five TRVs. Sequence analysis reveals that of the three proteins encoded by the TRV S1 genome segment, σC shares at most 57% amino acid identity with σC from the chicken reovirus reference strain S1133, while the most similar p10 and p17 proteins share 72% and 61% identity, respectively, with the corresponding S1133 proteins. The most closely related mammalian reovirus, the fusogenic Nelson Bay reovirus, encodes a σC protein that shares from 25% to 28% amino acid identity with the TRV σC proteins. This report supports the earlier suggestion that the TRVs are a separate virus species within the Orthoreovirus genus, and may provide some insight into TRV host specificity and pathogenesis. NC/SEP–R44/03 S1 genome segment DQ525419 NC/98 S1 genome segment DQ995806 TRV sigmaC sequences DQ996601–DQ996605 TRV p10 sequences DQ996606–DQ996610 TRV p17 sequences DQ996611–DQ996615     

Sequence and secondary structure analysis of the 5'-terminal region of flavivirus genome RNA

The 5'-terminal noncoding region sequences were determined for the genome RNAs of seven strains of St. Louis encephalitis virus (SLEV) and one strain of West Nile virus (WNV) using a single synthetic cDNA primer complementary to the 5'-terminus of the coding region of a strain of WNV RNA. The 5'-terminal sequences obtained for the SLEV and WNV RNAs were compared with published sequences for yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), and dengue virus. While only short regions within the 5'-noncoding sequence were conserved among different flavivirus RNAs, significant homology was observed in this region among members of the same flavivirus subgroup and almost complete conservation was observed between different strains of the same virus. For example, seven strains of SLE, isolated from different geographic locations over a 17-year period and differing in their neurovirulence phenotype, contained only two to four nucleotide changes in the 5'-noncoding region. Interestingly, each of three low-virulence strains shared the same unique base substitution at position 16. Secondary structures predicted to be formed by the 5'-termini of each of the different flavivirus genome RNAs were of similar size and shape, in each case consisting of a stem with a small top loop and a larger side loop. The prediction of a common structure among a number of different flaviviruses, despite the lack of extensive sequence homology, suggests that this secondary structure is functionally important. An additional stem and loop structure is predicted to be formed in the region spanning the translation initiation codon. This structure showed significantly less conservation of size and shape than the 5'-terminal secondary structure.     

Sequence and phylogenetic analysis of the large (L) segment of the Tahyna virus genome

The Tahyna virus (TAHV) is an important human pathogen in the Bunyaviridae family. To date, only the S and M segments of this virus have been sequenced, but the sequence of the L segment hasn’t been established yet. In this study, we sequenced 963 nucleotides of the L segment of TAHV, comprising pre-motif A and motif A in region 3 of the RNA polymerase gene. The nucleotide sequence data reported in this paper was submitted to the GenBank nucleotide sequence database: EU185046.