Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650 bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.

An ascovirus isolated from Spodoptera litura (Noctuidae: Lepidoptera) transmitted by the generalist endoparasitoid Meteorus pulchricornis (Braconidae: Hymenoptera).

The family Ascoviridae is a recently described virus family whose members are transmitted by parasitoids and cause chronic and lethal infections in lepidopteran insects. Little is known about the biology and ecology of ascoviruses, and few isolates have been found outside the United States. We report here the isolation of a new ascovirus variant from Spodoptera litura in Japan. Full genome sequence and phylogenetic analyses showed that this virus was closely related to variants in Heliothis virescens ascovirus-3a, and it was named HvAV-3j. HvAV-3j has a DNA genome of 191 718 bp, with 189 putative ORFs and a GC content of 45.6 %, and is highly similar to HvAV-3h, which was isolated in China. In a field survey, the endoparasitoid Meteorus pulchricornis caused a high percentage of parasitization in populations of S. litura larvae, and under laboratory conditions M. pulchricornis was able to transmit HvAV-3j from infected to uninfected larvae by oviposition. Meteorus pulchricornis is thus likely to be a major vector for HvAV-3j transmission in Japan. This species is recognized here for the first time as a vector of ascoviruses that parasitizes a range of host species that extends across families.

Genome analysis of Heliothis virescens ascovirus 3h isolated from China.

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.

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.

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.

Transcriptional analysis of a major capsid protein gene from Spodoptera exigua ascovirus 5a.

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.

Characterization of three ascovirus isolates from cotton insects.

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.