Xiao-Wen Cheng

Strategy of the use of 28S rRNA as a housekeeping gene in real-time quantitative PCR analysis of gene transcription in insect cells infected by viruses ☆

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.

P34.8 (GP37) is not essential for baculovirus replication

Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NOT:I site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NOT:I-XBA:I) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.

Phylogenetic Position and Replication Kinetics of Heliothis virescens Ascovirus 3h (HvAV-3h) Isolated from Spodoptera exigua

Insect-specific ascoviruses with a circular genome are distributed in the USA, France, Australia and Indonesia. Here, we report the first ascovirus isolation from Spodoptera exigua in Hunan, China. DNA-DNA hybridization to published ascoviruses demonstrated that the new China ascovirus isolate is a variant of Heliothis virescens ascovirus 3a (HvAV-3a), thus named HvAV-3h. We investigated the phylogenetic position, cell infection, vesicle production and viral DNA replication kinetics of HvAV-3h, as well as its host-ranges. The major capsid protein (MCP) gene and the delta DNA polymerase (DNA po1) gene of HvAV-3h were sequenced and compared with the available ascovirus isolates for phylogenetic analysis. This shows a close relationship with HvAV-3g, originally isolated from Indonesia, HvAV-3e from Australia and HvAV-3c from United States. HvAV-3h infection induced vesicle production in the SeE1 cells derived from S. exigua and Sf9 cells derived from S. frugiperda, resulting in more vesicles generated in Sf9 than SeE1. Viral DNA replication kinetics of HvAV-3h also demonstrated a difference between the two cell lines tested. HvAV-3h could readily infect three important insect pests Helicoverpa armigera (Hübner), Spodoptera exigua (Hübner) and Spodoptera litura (Fabricius) from two genera in different subfamilies with high mortalities.

Slow cell infection, inefficient primary infection and inability to replicate in the fat body determine the host range of Thysanoplusia orichalcea nucleopolyhedrovirus

Thysanoplusia orichacea multicapsid nucleopolyhedrovirus (ThorMNPV) carrying an enhanced green fluorescent protein (EGFP) gene expression cassette (vThGFP) was used to study host-range mechanisms. Infection kinetics showed that vThGFP replication in Sf21 cells was too slow to suppress cell growth. Wide-host-range Autographa californica MNPV (AcMNPV) could speed up vThGFP infection and enhance the vThGFP infection rate in Sf21 cells. The enhancement was not due to recombination, as no recombinant virus was isolated from co-infection by plaque assay. No improvement of vThGFP infection in Sf21 was found by AcMNPV cosmid transactivation assay. However, culture medium from Sf21 cells infected with AcMNPV did enhance vThGFP replication in Sf21. Third-instar larvae of Spodoptera frugiperda, S. exigua and Helicoverpa zea were not killed by feeding with vThGFP polyhedra but were killed by intrahaemocoelic injection using budded viruses (BVs). This suggested that insufficient BVs were generated during the primary infection in the midgut. vThGFP infected haemocytes, tracheae and Malpighian tubules but not fat bodies of larvae of S. frugiperda, S. exigua and H. zea. Third-instar S. frugiperda larvae co-infected by injection with vThGFP and vAcDsRed2, an AcMNPV expressing a red fluorescent protein gene, showed EGFP expression in the fat body. This result suggests that vAcDsRed2 could help vThGFP to replicate in the fat body or trans-activate EGFP expression in the fat body. All these results suggested that slow cell infection, insufficient primary infection and inability to replicate in the fat body control the host range of ThorMNPV.

Baculovirus interactions in vitro and in vivo.

Baculoviruses are promising viral insecticides and are safe for the environment. Interaction of baculoviruses in vitro and in vivo is a basic molecular and ecological question that has practical applications in agriculture. Cellular secretion is also a fundamental property in cell-cell communication. Here, we review recent investigations on how baculoviruses interact with insect cells and insect hosts. We focus particularly on a new interaction mechanism in which a secretion from cells infected with one virus enhances infection by a second virus. We also discuss a hypothesis that the secreted signals may serve as ligands that bind to the receptors on the surface of the cells that harbor the suppressed genomes of Thysanoplusia orichalcea MNPV (ThorMNPV) in Sf21 and Spodoptera exigua MNPV (SeMNPV) in High 5 to initiate signal transduction leading to the activation of genome replication of ThorMNPV in Sf21 and SeMNPV in High 5. We also discuss how the enhanced replication of SeMNPV replication by Autographa californica MNPV (AcMNPV) in nonpermissive insect cells depends on the types of cells. Interaction of baculoviruses in insects focused on mutualism and antagonism, even though the mechanism is not clear on mutualism. The antagonism of a Nucleopolyhedrovirus (NPV) with a Granulovirus (GV) has been extensively studied by a metalloprotein in the capsule of GV that disrupts the peritrophic membrane, a physical barrier to NPV entry to the midgut of larvae, to facilitate NPV infection.

Reduction of polyhedrin mRNA and protein expression levels in Sf9 and Hi5 cell lines, but not in Sf21 cells, infected with Autographa californica multiple nucleopolyhedrovirus fp25k mutants.

During cell infection, the fp25k gene of baculoviruses frequently mutates, producing the few polyhedra (FP) per cell phenotype with reduced polyhedrin (polh) expression levels compared with wild-type baculoviruses. Here we report that the fp25k gene of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), contains two hypermutable seven-adenine (A7) mononucleotide repeats (MNRs) that were mutated to A8 MNRs and a TTAA site that had host DNA insertions, producing fp25k mutants during Sf21 cell infection. The FP phenotype in Sf9 and Hi5 cells was more pronounced than in Sf21 cells. AcMNPV fp25k mutants produced similar levels of polyhedra or enhanced GFP, which were both under the control of the AcMNPV polh promoter for expression, in Sf21 cells but lower levels in Sf9 and Hi5 cells compared with AcMNPV with an intact fp25k gene. This correlated with the polh mRNA levels detected in each cell line. The majority of Sf21 cells infected with fp25 mutants showed high polh promoter-mediated GFP expression levels. Two cell lines subcloned from Sf21 cells that were infected with fp25k mutants showed different GFP expression levels. Furthermore, a small proportion of Hi5 cells infected with fp25k mutants showed higher production of polyhedra and GFP expression than the rest, and the latter was not correlated with increased m.o.i. Therefore, these data suggest that AcMNPV polh promoter-mediated gene expression activities differ in the three cell lines and are influenced by different cells within the cell line.

Development of Microplitis similis (Hymenoptera: Braconidae) on two candidate host species, Spodoptera litura and Spodoptera exigua (Lepidoptera: Noctuidae)

Abstract Microplitis similis Lyle (Hymenoptera: Braconidae) is a solitary endoparasitic braconid that generally parasitizes larvae of Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae) and many other noctuid species. To understand host preference, fitness, and the effects of M. similis on the hosts, we compared percentage parasitism, development periods, and the effects on host growth in candidate noctuid species. We found high levels of parasitism of S. exigua and Spodoptera litura (F.) larvae but not of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. The parasitoid wasp larvae took similar amounts of time for development on S. exigua and S. litura larvae, i.e., 13.87 ± 0.15 and 13.69 ± 0.42 d, respectively. Compared with the control larvae, the growth and development of the hosts were severely affected. The hosts were able to molt to 4th instars after being parasitized as early 3rd instars, but were unable to develop to the 5th instar. The body weight was similar between parasitized and non-parasitized larvae within the first 4 d (3 d in S. litura) but later began to show a significant difference from the 5th day on (4th day in S. litura). The host larvae eventually weighed up to 50 to 80% less than the non-parasitized larvae. Furthermore, the host larvae lived for an extended period in the same instar after egression of the parasitoid, but the body mass did not increase.

Using Host 28S Ribosomal RNA as a Housekeeping Gene for Quantitative Real‐Time Reverse Transcription‐PCR (qRT‐PCR) in Virus‐Infected Animal Cells

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. Curr. Protoc. Microbiol. 19:1D.2.1‐1D.2.13.

Genomic Sequence of Heliothis virescens Ascovirus 3g Isolated from Spodoptera exigua

ABSTRACT 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.

Comparative analysis of a highly variable region within the genomes of Spodoptera frugiperda ascovirus 1d (SfAV-1d) and SfAV-1a

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.