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The baculovirus-based bacmid expression vector system has been widely used for protein production in basic research and biotechnological laboratories. Since the first construction of the Autographa californica multiple nucleopolyhedrovirus bacmid (AcBacmid), three more bacmids have been created from Bombyx mori nucleopolyhedrovirus (BmBacmid), Spodoptera exigua nucleopolyhedrovirus (SeBacmid) and Helicoverpa armigera nucleopolyhedrovirus (HaBacmid). Each of these bacmid-derived viruses replicates efficiently in a range of specific and permissive cell types. Here, we investigated the relative stability of each virus derived from the bacmid during passage in permissive cell lines through assessment of their expression level and genome structure changes. Using two different reporters, the expression levels of the viruses from the AcBacmid-Sf9, AcBacmid-Tn5, BmBacmid-BmN and SeBacmid-SeE1 bacmid-cell systems were significantly reduced after five passages of the viruses, whereas the reductions were not detected in the AcBacmid-Sf21 and HaBacmid-HzAM1 systems. Pulse field gel electrophoresis (PFGE) and restriction fragment length polymorphism (RFLP) analysis of viral DNA isolated from passaged viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 systems showed no major genomic changes. In contrast, the genomes from passaged viruses in the AcBacmid-Tn5 and AcBacmid-Sf9 systems displayed reduced genome size and various mutations at individual loci, including genotypes missing one at least or more viral RNA polymerase subunits and fp25k. These genotypic changes were correlated with reduced protein expression. RFLP analysis of viral DNA from passaged viruses in the BmBacmid-BmN and SeBacmid-SeE1 systems exhibited changes in genome size, including excision of particular EcoRI fragments containing the mini-F replicon. Collectively, our data suggest that the viruses from the AcBacmid-Sf21 and HaBacmid-HzAM1 bacmid-cell systems are better for large-scale protein expression in continuous culture. Further study is needed to investigate the mechanism(s) behind the protein expression reduction in these bacmid-derived virus/cell systems.

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h (HvAV-3h) using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G+C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames (ORFs) along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 6a (TnAV-6a), and Diadromus pulchellus ascovirus 4a (DpAV-4a). Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs (bros) were present, and the typical homologous repeat regions (hrs) were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12250-016-3929-8 and is accessible for authorized users.

The complete genome of Trichoplusia ni ascovirus 6b (TnAV-6b) was sequenced for the first time. The TnAV-6b isolate, which has its closest phylogenetic relationship with the TnAV-6a isolate, has a circular genome of 185,664 bp, with a G+C content of 46.0% and 178 predicted open reading frames.

The complete genome sequence of Heliothis virescens ascovirus 3f (HvAV-3f) was obtained. The HvAV-3f genome has a circular genome of 198,157 bp with a G + C content of 46.0%, and encodes 190 open reading frames (ORFs) longer than 69 amino acids. Two major homologous regions ( hrs ) and 29 ‘baculovirus repeat ORFs’ ( bro ) were found in the genome. BLAST analyses revealed that three HvAV-3f genes were homologous to that of lepidopteran insects. Nine ORFs were unique to HvAV-3f, in which two ORFs showed significant levels of similarity to genes that have not been previously described for ascoviruses in the Genbank database.

Genome replication by the baculovirus DNA polymerase often generates errors in mononucleotide repeat (MNR) sequences due to replication slippage. This results in the inactivation of genes that affects different stages of the cell infection cycle. Here we mapped these MNRs in the 59 baculovirus genomes. We found that the MNR frequencies of baculovirus genomes are different and not correlated with the genome sizes. Although the average A/T content of baculoviruses is 58.67%, the A/T MNR frequency is significantly higher than that of the G/C MNRs. Furthermore, the A7/T7 MNRs are the most frequent of those we studied. Finally, MNR frequencies in different classes of baculovirus genes, such as immediate early genes, show differences between baculovirus genomes, suggesting that the distribution and frequency of different MNRs are unique to each baculovirus species or strain. Therefore, the results of this study can help select appropriate baculoviruses for the development of biological insecticides.

The major capsid protein (mcp) gene of Spodoptera exigua ascovirus 5a (SeAV-5a) was confirmed by aphidicolin viral DNA replication inhibition analysis to be a late gene. The 5' and 3' ends of mcp gene transcripts have been mapped. Primer extension analyses indicated that transcription of the mcp gene initiates from a cytosine 25 nucleotides (nt) upstream of the translation start codon. Two independent approaches by 3' rapid amplification of cDNA ends (3' RACE) and oligo (dT) cellulose binding assay suggested that SeAV-5a mcp mRNA is polyadenylated. Analyses by 3' RACE also revealed that mcp transcripts terminate at a U, either at 26 or 38 nt downstream of the translation stop codon. The putative 5' transcription control region of the SeAV-5a mcp gene shares similarities with other ascoviruses and Chilo iridescent virus (CIV), containing a conserved TATA-box-like motif (TAATTAAA) and an ATTTGATCTT motif upstream of it. The 3' downstream regions of the mcp gene of all the ascoviruses examined and CIV can form a stem-loop structure, and the ends of the mcp gene transcripts of SeAV-5a are within the predicted stem-loop region. This suggests that the stem-loop structure of the mcp gene might be involved in transcription termination.

Ascoviruses are a family of insect viruses with circular, double-stranded DNA genomes. With the sequencing of the Trichoplusia ni ascovirus 2c (TnAV-2c) genome, the virion structural proteins were identified by using tandem mass spectrometry. From at least eight protein bands visible on a Coomassie blue-stained gel of TnAV-2c virion proteins, seven bands generated protein sequences that matched predicted open reading frames (ORFs) in the genome, i.e. ORFs 2, 43, 115, 141, 142, 147 and 153. Among these ORFs, only ORF153, encoding the major capsid protein, has been characterized previously.

Ascoviruses, iridoviruses, asfarviruses and poxviruses are all cytoplasmic DNA viruses. The evolutionary origins of cytoplasmic DNA viruses have never been fully addressed. Morphological, genetic and molecular data were used to test if all four cytoplasmic virus families (Ascoviridae, Iridoviridae, Asfarviridae, and Poxvirirdae) evolved from nuclear replicating baculoviruses and how the four virus groups are related. Molecular phylogenetic analyses using DNA polymerase predicted that cytoplasmic DNA viruses might have evolved from nuclear replicating baculoviruses, and that poxviruses and asfarviruses share a common ancestor with iridoviruses. These three cytoplasmic viruses again shared a common ancestor with ascoviruses. Morphological and genetic data predicted the same evolutionary trend as molecular data predicted. A genome sequence comparison showed that ascoviruses have more baculovirus protein homologues than do iridoviruses, which suggested that ascoviruses have evolved from baculoviruses and iridoviruses evolved from ascoviruses. Poxviruses showed genetic and morphological similarity to other cytoplamic viruses, such as ascoviruses, suggesting it has undergone reticulate evolution via hybridization, recombination and lateral gene transfer with other viruses. Within the ascovirus family, we tested if molecular phylogenetic analyses agree with biological inference; that is, ascovirus had an evolutionary trend of increasing genome size, expanding host range and widening tissue tropism for these viruses. Both molecular and biological data predicted this evolutionary trend. The phylogenetic relationship among the four species of ascovirus was predicted to be that TnAV-2 and HvAV-3 shared a common ancestor with SfAV-1 and the three virus species again shared a common ancestor with DpAV-4.

The complete Trichoplusia ni ascovirus 2c (TnAV-2c) genome sequence was determined. The circular genome contains 174,059 bp with 165 open reading frames (ORFs) of greater than 180 bp and two major homologous regions (hrs). The genome is quite A+T rich at 64.6%. Fifty-four ORFs had homologues in other insect viruses, such as ascoviruses, iridoviruses, baculoviruses and entomopoxviruses; 30 ORFs showed low identities with those from different parasitic protozoa and 12 ORFs were unique to TnAV-2c. TnAV-2c has 15 ORFs that could be grouped into six gene families. Three major conserved repeating sequences were identified and were interspersed in two regions. BLAST analyses revealed that there were 16 enzymes involved in gene transcription, DNA replication, and nucleotide metabolism. TnAV-2c has 12 and 25 ORFs sharing high identities with ascovirus and iridovirus homologues, respectively. The codon usage bias appears to be more similar to Spodoptera frugiperda ascovirus 1a than to iridoviruses.

The nucleocytoplasmic large DNA viruses (NCLDVs) comprise a monophyletic group of viruses that infect animals and diverse unicellular eukaryotes. The NCLDV group includes the families Poxviridae, Asfarviridae, Iridoviridae, Ascoviridae, Phycodnaviridae, Mimiviridae and the proposed family "Marseilleviridae". The family Mimiviridae includes the largest known viruses, with genomes in excess of one megabase, whereas the genome size in the other NCLDV families varies from 100 to 400 kilobase pairs. Most of the NCLDVs replicate in the cytoplasm of infected cells, within so-called virus factories. The NCLDVs share a common ancient origin, as demonstrated by evolutionary reconstructions that trace approximately 50 genes encoding key proteins involved in viral replication and virion formation to the last common ancestor of all these viruses. Taken together, these characteristics lead us to propose assigning an official taxonomic rank to the NCLDVs as the order "Megavirales", in reference to the large size of the virions and genomes of these viruses.

Bombyx mori nucleopolyhedrovirus (BmNPV), a member of the Baculoviridae , is a major pathogen of silkworm and has also been recently developed as an expression vector for heterologous gene expression in the silkworm larvae and pupae. To better understand the diversity of this important baculovirus, we sequenced the complete genome of the BmNPV strain isolated from India, where its host is available throughout the year due to its tropical climate. The genome of the Indian strain consists of 127,879 nucleotides, with a G+C content of 40.36%. There are 138 open reading frames (ORFs) encoding the predicted proteins of more than 50 amino acids. Genomic comparison of the Indian strain with 3 other reported BmNPV strains showed that the baculovirus repeat ORFs ( bro ) and homologous repeat regions (hr's) are highly variable. These results suggest that the BmNPV strain heterogeneity is mainly caused by single-nucleotide polymorphisms (SNPs) and changes in the hr's and bro genes.

The recently discovered ascoviruses have a worldwide distribution. Here we report a new member of the family Ascoviridae, Spodoptera frugiperda ascovirus 1d (SfAV-1d) with a variable region in the genome. Restriction fragment length polymorphism, Southern hybridization and genome sequencing analyses confirmed that SfAV-1d and the earlier reported SfAV-1a are closely related but are not identical. The genome size of SfAV-1d is approximately 100 kbp, which is about 57 kbp smaller than SfAV-1a. The SfAV-1d genome has a major deletion of 14 kbp that corresponds to one of the inverted repeat (IR) regions of SfAV-1a. Cloning and sequencing revealed that the region flanking the deletion within the SfAV-1d genome is highly variable. In all the variants of this region, the whole IR region is missing, with 88.2 % of the variants missing part of or the whole adjacent SfAV-1a ORF71, 94.1 % missing part of or the whole of adjacent ORF72 and 64.6 % missing part of or the whole of ORF73.

The use of quantitative real-time reverse transcription-PCR (qRT-PCR) for studying regulation of gene transcription requires an internal template-loading control or a housekeeping gene to guarantee the validity of the data collection, analysis, and interpretation. Analysis of gene transcription in virus-infected animal cells is problematic because virus infection often results in modified or fluctuating gene transcription patterns of conventionally used housekeeping genes, such as the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene and the β-actin gene. It has been demonstrated that the host 28S ribosomal gene can be used as a housekeeping gene in qRT-PCR in virus-infected insect cells. The stability of the human 28S rRNA gene transcription during the infection of HeLa cells with adenovirus has been confirmed, and this method has been extended to the use of the human 28S rRNA gene as a housekeeping gene in adenovirus-infected HeLa cells. Step-by-step instructions are described for use of this control in analysis of gene transcription in both types of virus-infected animal cells.

Quantitative real-time reverse transcription-PCR (qRT-PCR) has been used widely to measure gene transcription regulation in cells. qRT-PCR must include one or more internal housekeeping genes to normalize data collection. A strategy to use the host cell 28S rRNA as a housekeeping gene in qRT-PCR analysis of gene transcription of insect cells infected by baculovirus and ascovirus was developed. It has been found that the 28S rRNA reverse primer can be incorporated in the oligo-dT-primed cDNA synthesis reaction. In such a way, amplification of 28S cDNA showed lower and less variable cycle thresholds in cells infected by viruses than by using only oligo-dT and other published housekeeping genes such as the TATA box binding protein (TBP) gene, the peptidyl prolyl isomerase A (PPI) gene and the ribosomal protein 13 (L13) gene. Incorporation of the 28S reverse primer in oligo-dT-primed cDNA synthesis also does not interfere with the detection of other polymerase II transcribed genes.

Three new ascovirus isolates were discovered from lepidopteran larvae in cotton fields in Blackville, South Carolina, USA, and were named TnAV-2c, TnAV-2d, and HvAV-3f. TnAV-2c and TnAV-2d were compared by restriction endonuclease (REN) profiles and found to be similar. HvAV-3f was isolated from Helicoverpa zea, and bears remarkable dissimilarity in REN profiles to the reported SeAV-5a from Spodoptera exigua but DNA hybridization shows they are closely related. Major capsid protein (MCP) and delta DNA polymerase from the three isolates were sequenced, which suggests the three isolates are novel. Phylogenetic analyses showed that TnAV-2c is distantly related to other lepidopteran ascoviruses. HvAV-3f and SeAV-5a may also be variants of the same species based on Southern, Western, and MCP/DNA polymerase gene sequence analyses. High levels of TnAV-2 infection in an H. zea population (as high as 74%) were recorded in a cotton field in Blackville, SC. Observations in this field showed that infection by ascovirus altered the feeding behavior of H. zea larvae.

Heliothis virescens ascovirus 3a (HvAV-3a), a member of the family Ascoviridae , has the highest diversity among ascovirus species that have been reported in Australia, Indonesia, China, and the United States. To understand the diversity and origin of this important ascovirus, the complete genome of the HvAV Indonesia strain (HvAV-3g), isolated from Spodoptera exigua , was determined to be 199,721 bp, with a G+C content of 45.9%. Therefore, HvAV-3g has the largest genome among the reported ascovirus genomes to date. There are 194 predicted open reading frames (ORFs) encoding proteins of 50 or more amino acid residues. In comparison to HvAV-3e reported from Australia, HvAV-3g has all the ORFs in HvAV-3e with 6 additional ORFs unique to HvAV-3g, including 1 peptidase C26 gene with the highest identity to Drosophila spp. and 2 gas vesicle protein U (GvpU) genes with identities to Bacillus megaterium . The five unique homologous regions (hrs) and 25 baculovirus repeat ORFs (bro) of HvAV-3g are highly variable.

Thysanoplusia orichalcea multiple nucleopolyhedrovirus (ThorMNPV) has high virulence to Trichoplusia ni and Pseudoplusia includens larvae, with a potential for biological control of insect pests. The genome of ThorMNPV was sequenced and found to be 132,978 bp, with a G+C content of 37.9%. There are 145 predicted open reading frames (ORFs), encoding proteins of 50 or more amino acid residues with minimal overlap. Of the 145 ORFs, 141 appeared to be homologous to those of Autographa californica MNPV (AcMNPV). In comparison to AcMNPV, 9 ORFs of AcMNPV were absent in ThorMNPV, including the superoxide dismutase ( sod ) gene.