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.
Citation: Guo-Hua Huang, Dian-Hai Hou, Manli Wang, Xiao-Wen Cheng, Zhihong Hu. Genome analysis of Heliothis virescens ascovirus 3h isolated from China[J]. VIROLOGICA SINICA, 2017, 32 (2): 147-154 https://doi.org/10.1007/s12250-016-3929-8
Received: 8 December, 2016; Accepted: 15 February 2017; Published: 30 March 2017
Copyright: © Wuhan Institute of Virology, CAS and Springer Science+Business Media Singapore 2017
Data Availability: All relevant data are within the paper and its Supporting Information files.
Corresponding author: Phone: +86-27-87197180, Fax: +86-27-87197180, Email: firstname.lastname@example.org ORCID: 0000-0002-1560-0928.
Ascoviridae is an insect-specific family of viruses with double-stranded circular DNA genomes of 110–200 kb (Bigot et al., 2011; Wei et al., 2014). Ascoviruses can cause a chronic but ultimately fatal disease in the larvae of members of the Noctuidae, Crambidae, and Plutellidae families (Bigot et al., 2011). Based on virion morphology, DNA sequence information, host range, and tissue tropism, two genera, Ascovirus and Toursvirus, have been recognized by the International Committee on Taxonomy of Viruses (ICTV) (Asgari et al., 2017). The genus Ascovirus contains three species–Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 2a (TnAV-2a), and Heliothis virescens ascovirus 3a (HvAV-3a) (Bigot et al., 2011)–while the newly assigned genus Toursvirus consists of only one species, Diadromus pulchellus ascovirus 4a (DpAV-4a), that was recently removed from the genus Ascovirus (Asgari et al., 2017).
A total of 18 ascoviral isolates or strains have been reported globally so far (Bigot et al., 2011; Huang et al., 2012a). Among these, the genomes of only 6 isolates have been sequenced: SfAV-1a (Bideshi et al., 2006), TnAV-6a (previously named as TnAV-2c, Wei et al., 2014) (Wang et al., 2006), HvAV-3e (Asgari et al., 2007), HvAV-3f (Wei et al., 2014), HvAV-3g (Huang et al., 2012b), and DpAV-4a (Bigot et al., 2009). Among the sequenced isolates, HvAV-3g contains the largest genome (199, 721 bp), while DpAV-4a has the smallest one (119, 343 bp) (Bigot et al., 2009; Huang et al., 2012b).
Based on the analysis of biological characteristics, the species HvAV-3a, which consists of 8 isolates, has been found to be the most diverse and widely distributed species from America to Asia and Australia (Hamm et al., 1998; Huang et al., 2012a). Among HvAV-3a species, the genome sequences of 3 isolates–HvAV-3e, HvAV-3g, and HvAV-3f–have been reported from Australia, Indonesia, and USA, respectively (Asgari et al., 2007; Huang et al., 2012b; Wei et al., 2014). However, no ascovirus isolated from China has been sequenced so far. To understand the diversity and phylogeny of HvAV-3a isolates, we aimed to sequence the complete genome of a HvAV-3h, which was isolated from China and transmitted by Microplitis similis (Li et al., 2016). We then went on to compare the sequence with the other 6 previously published ascovirus genome sequences. The results of our study will be helpful for providing insights into the origin and evolution of ascoviruses.
Viral DNA extraction
For this study, HvAV-3h was propagated in Spodoptera exigua larvae as described previously (Huang et al., 2012a). The virions were then purified and viral DNA was extracted as previously described (Federici et al., 1990).
Sequencing and bioinformatics analysis
The genome was sequenced with the Roche 454 GS FLX system by using shotgun strategy. The reads were assembled with Roche GS De Novo assembler software. A few regions that were not assembled into contigs were further amplified using PCR and sequenced.
Hypothetical ORFs were predicted using ORF finder (NCBI) with characteristics of containing a standard ATG start codon and a stop codon and potentially encoding at least 50 amino acids (aa). Gene annotation and comparisons were performed using the NCBI protein-protein BLAST algorithm (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Homologies among ascovirus genomes were investigated using Basic Local Alignment Search Tool (BLAST) (blastp) in NCBI (Altschul et al., 1997) and two sequences were aligned using blastp. MIROPEATS program (Parsons, 1995) was used to find repetitive regions. Restriction sites were predicted and the genome map framework was drawn using the program Geneious vesion 8.04. The annotated genome sequence data were uploaded to GenBank under the accession number KU170628.
Phylogenetic analysis of the ascoviruses was performed using the amino acid sequences of DNA polymerase and Major capsid protein (MCP) from HvAV-3h and the 6 other sequenced Ascovirus and Toursvirus isolates, with homologs from invertebrate iridescent virus 22 and Wiseana iridescent virus as the outgroups (Piégu et al., 2015). The sequences were then aligned using ClustalW (Larkin et al., 2007) with default parameters of MEGA 6.0 (Tamura et al., 2013). The maximum likelihood (ML) method was used with the program raxmlGUL 1.5 (https://sourceforge.net/projects/raxmlgui/) (Silvestro and Michalak, 2012), in which the analysis setting was chosen “ML+rapid bootstrap” and the “number of bootstrap replicates” was 1, 000. The program MrBayes 3.1.2 (http://morphbank.Ebc.uu.SE/mrbayes/) was used to perform Bayesian inference (BI) analysis (Ronquist et al., 2003), with the MCMC analysis run for 300,000 generations and a burn-in series of 1,000.
General organization of the HvAV-3h genome
The HvAV-3h genome was sequenced using the Roche 454 GS FLX system with the shotgun strategy. A total of 43, 218 reads were obtained with the average length of 423 bp, and the genome was covered about 53 times. The HvAV-3h genome was assembled using Roche GS De Novo assembler software and Geneious version 8.04. A few regions that were not assembled into contigs were further amplified using PCR with the primers listed in Supplementary Table S1, cloned, and sequenced. The final assembled contig representing the entire HvAV-3h genome sequence was confirmed using restriction digestion with 6 enzymes (BamH I, Hind III, Nde I, Pst I, Sac I, and Xho I). The restriction profile (Supplementary Figure S1) matched well with the predicted fragments of the assembled genome (Supplementary Table S2). However, there were still some submolar bands (Supplementary Figure S1), indicating the presence of different genotypes in the isolate.
The HvAV-3h genome was assembled into a circular contiguous sequence of 190, 519 bp, which was significantly larger than the genome size (165 kbp) estimated previously (Huang et al., 2012a). This was possibly largely due to the missing small size fragments in the previously reported restriction enzyme digestion profiles (Huang et al., 2012a). A genomic map showing the organization of the ORFs with the predicted protein of over 50 aa in the HvAV-3h genome is presented in Figure 1. The genome has a G+C content of 45.5% and encodes 185 predicted ORFs, in which 104 are in the forward orientation and 81 are in the reverse orientation (Figure 1). The coding region accounts for 88.8% of the total sequences.
Relationship with other ascoviruses
The size of the HvAV-3h genome was found to be smaller than that of the HvAV-3g (199, 721 bp) and HvAV-3f genomes (198, 157 bp), but larger than that of the HvAV-3e genome (186, 262 bp). It is thus the third largest ascovirus genome described so far. Gene-parity plots of HvAV-3h against the 6 ascovirus isolates revealed high co-linearity of the gene order between HvAV-3h and HvAV-3g, HvAV-3f, or HvAV-3e, with only a few inversions and drifts, which may partially account for the difference in the genome size. Much lower co-linearity was found between HvAV-3h and SfAV-1a or TnAV-6a. No obvious collinear region could be found between HvAV-3h and DpAV-4a (Figure 2). The collinear regions between HvAV-3h and other ascovirus isolates, with the exception of DpAV-4a, indicated the presence of conserved regions among ascoviral genomes and their derivation from an ancestor ascovirus.
Phylogenetic analyses of the conserved genes of DNA polymerase and MCP from HvAV-3h and the 6 other Ascovirus and Toursvirus isolates also revealed a close relationship between HvAV-3h, HvAV-3g, HvAV-3f, and HvAV-3e (Figure 3). In fact, analysis using ML and Bayesian methods with a bootstrap value of 100% and a posterior probability of 1.00, respectively, revealed that these four HvAV-3a isolates form a monophyletic clade at high levels. The HvAV-3a isolates also appeared to have a distant relationship with SfAV-1a and TnAV-6a and an even more distant relationship with DpAV-4a (Figure 3).
Comparison and classification of gene contents of the HvAV-3h genome with that of other ascoviruses
BLAST analysis revealed that, among the identified ORFs, 181 ORFs were related to genes reported from the other 6 ascovirus isolates, including HvAV-3g (174 ORFs, with average amino acid identity of 94.0%), HvAV-3f (172 ORFs, 92.9%), HvAV-3e (162 ORFs, 87.5%), SfAV-1a (116 ORFs, 62.7%), TnAV-6a (105 ORFs, 56.7%), and DpAV-4a (63 ORFs, 34.0%) (Supplementary Table S3).
Forty-four ascovirus ORFs were found to be conserved among HvAV-3h and the other 6 ascovirus and toursvirus genomes. Gene annotation indicated that these genes are involved in DNA/RNA replication/transcription/metabolism, viral packaging and assembly, sugar and lipid metabolisms, etc. (Table 1). All the ascovirus isolates except DpAV-4a shared 25 ORFs with HvAV-3h (Table 2). Some of them appear to play roles in DNA/RNA replication/transcription/metabolism, but the functions of most of the shared genes are still unknown (Table 2). A total of 159 genes including the above-mentioned genes were found to be shared between HvAV-3h and the three closely related isolates of the species HvAV-3a. In addition, 19 genes were found to be shared between HvAV-3h and any one but not all three previously reported isolates of the species HvAV-3a. Six ORFs including ORF49 (GIY-YIG-like endonuclease), ORF51, ORF86, ORF92, ORF96, and ORF159 were not found in any of the other three isolates. ORF49 showed significant similarity to GIY-YIG-like endonuclease with E-value of 6e-46. GIY-YIG-like endonuclease was found in SfAV-1a, TnAV-6a, and DpAV-4 as well as in viruses of the Baculoviridae and Iridoviridae families. Homologues of GIY-YIG-like endonuclease are known to be involved in viral DNA repair (Lindahl, 1982) and are required for efficient baculoviral virion production (Tang et al., 2013; Wu & Passarelli, 2012). ORF51 with an unknown function only showed significant similarity to ORF39 of SfAV-1a with E-value of 1e-44. The other four ORFs, including ORF86, ORF92, ORF96, and ORF159, showed no significant levels of similarity to genes in the GenBank database; hence, they were considered unique genes of HvAV-3h.
The baculovirus repeat ORF (bro) gene occurs as multiple copies per ascovirus genome (Asgari et al., 2007). For example, the genomes of SfAV-1a, TnAV-6a, HvAV-3e, HvAV-3f, HvAV-3g, and DpAV-4a show 7, 3, 23, 29, 25 and 12bro genes, respectively (Asgari et al., 2007; Bideshi et al., 2006; Bigot et al., 2009; Huang et al., 2012b; Wang et al., 2006; Wei et al., 2014). In our study, we found that HvAV-3h contains 24bro genes that encode from 99 to 521 residues (Figure 1, Supplementary Table S3). The bro genes in baculovirus have been believed to help the virus acquire a new functionality eventually (de Castro Oliveira et al., 2013), but their function in ascoviruses remains to be determined.
HvAV-3h is the fourth sequenced isolate of the species HvAV-3a. HvAV-3h had a similar gene order and shared 159 ORFs with all the other isolates of the species HvAV-3a (Figure 2, Supplementary Table S3). However, there are still some gene reversions and drifts among the genomes. For example, 19 ORFs were found in any one but not all the four isolates, and 6 ORFs were only found in HvAV-3h (Figure 2, Supplementary Table S3). These findings show that although HvAV-3a isolates are closely related to each other, there are noticeable diversities in their genome organizations.
Comprehensive Geneparity Plots and phylogeny analyses of all the sequenced Ascoviridae isolates indicated that HvAV-3a isolates are distantly related to SfAV-1a and TnAV-6a and most distantly to DpAV-4a (Figure 2, Figure 3). All the Ascoviridae isolates shared 44 conserved genes (Table 1), which likely play important roles during the life cycle of the viruses. Among the 44 conserved ORFs, 16–DNA polymerase (orf1), RNA polymerase subunits (orf9, orf61, orf80, and orf143), DEAD-like helicase (orf13), RNase III (orf24), poxvirus late transcription factor VLTF3 like protein (orf30), thymidine kinase (orf52), DNA repair exonuclease (orf67), ATPase involved in DNA metabolism (orf116), CDT phosphatase transcription factor (orf117), ATPase involved in DNA repair (orf125), ATPase involved in DNA replication (orf126), yabby-like transcription factor (orf135), and tyrosyl-DNA phosphodiesterase (orf157)–are predicted to be involved in DNA/RNA replication/transcription and metabolism (Table 1). All the ascoviruses appear to encode many genes involved in nucleotide metabolism and transcription/replication. For example, TnAV-6a encodes 16 related ORFs (Wang et al., 2006) and SfAV-1a encodes 9 (Bideshi et al., 2006).Serine/threonine protein kinase and AV-like serine/ threonine protein kinase are also conserved in ascoviruses. These kinases likely participate in virus or virus-host regulation of signaling networks (Jacob et al., 2011); however, the roles of the kinases in ascovirus infection are not clear so far. Efforts also need to be made to reveal the functions of other unknown conserved ORFs (Table 1). Among the 44 core genes, only 2 were annotated to encode the structural proteins MCP (orf53) and sulfhydry1 oxidase Erv1-like protein (orf71) (Long et al., 2009; Wu and Passarelli, 2010). As ascoviruses have complicated morphologies, it is tempting to assume that more structural genes may be identified from the remaining unknown core genes.
Ascovirus genomes often contain large interspersed repeats of 1–3 kbp (Bigot et al., 2000), but no repeat regions (hrs) were found in the HvAV-3h genome (Figure 1). Similarly, no repeat regions have been found in the DpAV-4a genome (Bigot et al., 2009). In contrast, 5 copies of repeats have been found in the HvAV-3e genome (orf38 <hr1>, orf84 <hr2>, orf124 <hr3>, orf156 <hr4> and orf178 <hr5>) (Asgari et al., 2007) and the HvAV-3g genome (orf35, orf68, orf104, orf126, and orf173) (Huang et al., 2012b). Two repeat regions have been found in the HvAV-3f genome (orf95 and orf179) (Wei et al., 2014) and the SfAV-1a (orf34 and orf77) (Bideshi et al., 2006) and TnAV-6a (Hr1 and Hr2) genomes (Wang et al., 2006). Additionally, 24bros were found in the HvAV-3h genome in our study, and 12, 7, 3, 23, 26 and 29bros have been found previously in DpAV-4a (Bigot et al., 2009), SfAV-1a (Bideshi et al., 2006), TnAV-6a (Wang et al., 2006), HvAV-3e (Asgari et al., 2007), HvAV-3g (Huang et al., 2012b), and HvAV-3f (Wei et al., 2014), respectively. The function of repeat regions, bros, and hrs in the evolution and variety of ascovirus genomes needs to be investigated in future studies.
In summary, we sequenced the genome of a HvAV-3h isolate from China and compared the sequence to those of 6 other previously published ascovirus genome sequences to establish the evolutionary relationship between different ascovirus species. Our findings indicate a close relationship between different HvAV-3a isolates and distant relationship between HvAV-3h and some other species of the Ascoviridae family. Future studies need to be focused on elucidating the diversities in the genome organizations of different HvAV-3h isolates and on understanding the role of different genes in the ascoviral genomes as well as the conservation of these genes.
The authors would like to thank Jue Hu, Xing-Shi Gu, Wen-Fei Xian, Hai-Zhou Liu, Lei Zhang, and Shun-Ji Li for their help in virus amplification and data analysis. The authors would also like to acknowledge the technical assistance received from the Core Facility and Technical Support of Wuhan Institute of Virology. This study was supported partly by the National Natural Science Foundation of China (No. 31371995 and 31621061), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant XDB11030400), STS Project of the Chinese Academy of Sciences (grant KFJ-SW-STS-143-3), and the Hunan Provincial Natural Science Foundation for Distinguished Young Scholar of China (14JJ1023).
COMPLIANCE WITH ETHICS GUIDELINES
The authors declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors.
Conceived and designed the experiments: HGH, DHH, MW, XWC, ZH. Performed the experiments: HGH, DHH. Analyzed the data: HGH, DHH, ZH. Contributed reagents/materials/analysis tools: HGH, DHH. Wrote the paper: HGH, DHH, ZH.
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