Analysis of the genomic sequence of Philosamia cynthia nucleopolyhedrin virus and comparison with Antheraea pernyinucleopolyhedrin virus
© Qian et al; licensee BioMed Central Ltd. 2013
Received: 17 October 2012
Accepted: 31 January 2013
Published: 20 February 2013
Two species of wild silkworms, the Chinese oak silkworm (Antheraea pernyi) and the castor silkworm Philosamia cynthia ricini, can acquire a serious disease caused by Nucleopolyhedrin Viruses (NPVs) (known as AnpeNPV and PhcyNPV, respectively). The two viruses have similar polyhedral morphologies and their viral fragments share high sequence similarity. However, the physical maps of the viral genomes and cross-infectivity of the viruses are different. The genome sequences of two AnpeNPV isolates have been published.
We sequenced and analyzed the full-length genome of PhcyNPV to compare the gene contents of the two viruses. The genome of PhcyNPV is 125, 376 bp, with a G + C content of 53.65%, and encodes 138 open reading frames (ORFs) of at least 50 amino acids (aa) (GenBank accession number: JX404026). Between PhcyNPV and AnpeMNPV-L and -Z isolates, 126 ORFs are identical, including 30 baculovirus core genes. Nine ORFs were only found in PhcyNPV. Four genes, cath, v-chi, lef 10 and lef 11, were not found in PhcyNPV. However, most of the six genes required for infectivity via the oral route were found in PhcyNPV and in the two AnpeNPV isolates, with high sequence similarities. The pif-3 gene of PhcyNPV contained 59 aa extra amino acids at the N-terminus compared with AnpeNPV.
Most of the genes in PhcyNPV are similar to the two AnpeNPV isolates, including the direction of expression of the ORFs. Only a few genes were missing from PhcyNPV. These data suggest that PhcyNPV and AnpeNPV might be variants of each other, and that the differences in cross-infection might be caused by gene mutations.
Baculoviridae is a large family of viruses that infect and kill insect species of different orders. Worldwide, they have been reported to infect over 600 host species , mostly from the order Lepidoptera. However, the viruses also infect insects from the orders Diptera, Hymenoptera and the crustacean order, Decapoda.
The complete genomes of 57 baculoviruses have been deposited in GenBank, including 41 Alphabaculoviruses, 12 Betabaculoviruses, three Gammabaculoviruses and one Deltabaculovirus genome . Baculoviruses have been used extensively in many biological applications, for example as protein expression systems, as models of genetic regulatory networks and genome evolution, as putative nonhuman viral vectors for gene delivery, and as biological control agents against insect pests [4–6]. However, the diseases caused by baculoviruses are a major threat to the silk industry .
Silkmoths mostly belong to two families, the Bombycidae and Saturniidae, which secrete several varieties of silk fibers. The most common breeds are the domesticated silkworm (Bombyx mori L.) and the wild silkworms, including Chinese oak silkworm (Antheraea pernyi Guérin-Meneville), the castor silkworm (Philosamia cynthia ricini), the Indian tropical tasar silkworm (A. mylitta Drury) and the Japanese oak silkworm (A. ayamamai Guérin-Meneville) . Silk production by these moths, especially B. mori, A. pernyi and P. cynthia ricini, are economically important worldwide. The domesticated silkworm (B. mori) has been used for silk production by Chinese farmers for approximately 5000 years . It has since spread to Korea, Japan, India, Brazil and the rest of the world. The most well-known species among wild silkworms is the Chinese oak silkworm (A. pernyi). It is commercially cultivated for silk production, primarily in China, India and Korea . This silkworm species and the castor silkworm (P. Cynthia ricini) were introduced into China for silk production in the 1950s .
These three species can be infected by baculoviruses: B. mori Nucleopolyhedrosis virus (BmNPV), A. pernyi NPV (AnpeNPV) and P. Cynthia ricini NPV (PhcyNPV) according to their respective hosts [9–12]. The complete sequence of BmNPV strain T3 (GenBank: NC_001962) was published in 1999, and the sequence of B. mandarina NPV (BomaNPV, a variant of BmNPV) was published in 2010 (GenBank: FJ882854) [13, 14]. Two AnpeNPV isolates were published in 2007 (GenBank: EF207986 and NC_008035). We compared AnpeNPV and PhcyNPV, and found that these two viruses have similar polyhedral morphologies and their viral fragments show high sequence similarity. However, the physical maps of the viral genomes and cross-infectivity of the viruses are different. Therefore, we analyzed the whole genome sequence of PhcyNPV to obtain more information about the viral genes related to infection.
Sequencing, assembly, and analysis of the PhcyNPV genome
The entire PhcyNPV dsDNA genome was sequenced and assembled into a contiguous sequence of 125, 376 bp, with a G + C content of 53.65% (GenBank: JX404026). Many baculoviruses have an approximate GC content of 41%, whereas PhcyNPV and several other baculoviruses have significantly higher values (50.1% for CfMNPV, 50.9% for CuniNPV, 53.5% for AnpeNPV-L2, 53.5% for AnpeNPV-Z, 53.5% for LyxyNPV, 55.1% for OpMNPV and 57.5% for LdMNPV). However, a detailed analysis of DNA content did not show a clear pattern of GC content that could be associated with each genus .
Comparison of the PhcyNPV genome with the genomes of two AnpeNPV isolates
Certain ORFS were truncated or extended. For example, the p94 (Ac 134) genes in AnpeNPV-L2 and in PhcyNPV were predicted to encode proteins of 492 aa and 795 aa, respectively, whereas, in the –Z isolate, the gene could be divided into two ORFs (390 aa and 99 aa). Homologs of p94 are present in the genomes of most Group I baculoviruses. They are also present in some members of Group II and GV, and in several polydnaviruses. Disruption of the p94 gene had no effect on the ability of AcMNPV to infect S. frugiperda larvae by either the oral or intrahemocelic route . Interestingly, the egt genes of these three viruses are different; the ORF of PhcyNPV egt comprises 87 aa in the EGT N-terminus of AcMNPV, similar to AnpeNPV (−L2, 132 aa; -Z, 79 aa). The egt gene encodes ecdysteroid UDP-glucosyltransferase (EGT), and homologs are found in all Groups I, II and most GV genomes, mostly encoding proteins of 400 to 500 aa. The function of EGT is to block molting and pupation in infected larvae by catalyzing the transfer of glucose from UDP-glucose to ecdysteroids, thereby inactivating these insect molting hormones [16, 17]. Deletion of the egt gene from the genome of AcMNPV caused the virus to kill the insects more quickly . The day after P. cynthia ricini larvae were infected with PhcyNPV or AnpeNPV by per os inoculation, nearly one-third of the insects died. This might have been caused by egt mutation. The observation requires further investigation.
Predicting cellular location of the PhcyNPV genes
PhcyNPV ORFs with a predicted subcellular location in the ER (endoplasmic reticulum)
chitin-binding protein 1
PhcyNPV ORFs with a predicted mitochondrial location
EXO III v-trex
Of the eight unique predicted genes of PhcyNPV, five might be related to energy metabolism and/or apoptosis, and one is located in ER. These data imply that the unique genes of baculovirus mostly determine their interaction with individual insect breeds.
The genes related to oral infectivity
A previous report showed that the cross-infectivity characteristics of PhcyNPV and AnpeNPV were different. AnpeNPV caused 57% mortality in larvae of P. cynthia rici, whereas PcrNPV did not kill the larvae of A. pernyi. Hence, the six genes (pif 0 to 5) required for infectivity via the oral route were analyzed. Most of them are very similar between PhcyNPV and the two AnpeNPV isolates, except for PIF-3, which in PhcyNPV has a 59aa extra amino acid sequence in the N-terminus.
We observed that PhcyNPV was not infectious to A. pernyi by oral inoculation. This might involve the oral infection factors, including p74/ pif-0, pif-1, pif-2, pif-3, pif-4/19 k/odv-e28, and pif-5/odv-e56[25, 26]. These six genes are all structural components of occlusion derived viruses (ODVs). P74 mediates the specific binding of ODVs to primary cellular targets in the midgut epithelia , while pif-3 appears to mediate another crucial, but as yet unidentified, event during primary infection . The proteins encoded by pif-4 and pif-5 have essential roles in the per os infection route [24–26]. Most of these genes are highly similar to those of AnpeNPV, except for PIF-3 of PhcyNPV, which has an extra 59-aa N-terminal structure. However, no homologous structure was found in other viruses or species. The PIF-3 proteins from other baculoviruses are all about 200 aa, and show low levels of sequence similarity to each other. Interestingly, the C-terminal structure of PhcyNPV PIF-3 is similar to that of a membrane-spanning Ca-ATPase from the fungus Spathaspora passalidarum (GenBank: EGW31824), and an ABC transporter exported protein from the bacterium Pseudomonas sp. R81 (GenBank: ZP_11190693). These proteins are associated with the cell membrane. These observations imply that baculovirus PIF-3 might be related to the viral trans-membrane process.
The hrf1 gene from LdMNPV expands the host range of AcMNPV both in vitro and in vivo, allowing it to infect non-permissive hosts [27, 28]. The only baculovirus homolog of this gene is found in OpMNPV. Two conotoxin-like (ctl) genes, ctl1 and ctl2, are also found in both of these genomes. Other baculoviruses encode one or the other, but only OpMNPV and LdMNPV encode both. A report indicated that there is a clear phylogenetic link between hrf1and the presence of both ctl genes . We also found the two ctl genes in the genomes of PhcyNPV and AnpeNPV. However, the hrf1 gene was missing. It seems that functions of ctl1 and ctl2 are distinct when appearing with hrf1.
Most of the genes in PhcyNPV were similar to the two AnpeNPV isolates, including the direction of expression of the ORFs. Only a few genes were missing in PhcyNPV. These data suggest that PhcyNPV and AnpeNPV might be variants with each other, and the difference of cross-infection might be caused by gene mutations.
The Institute of Guangxi Sericultural Research and Development in South China provided the occlusion bodies (OBs) of PhcyNPV. The AnpeNPV was kindly provided by Professor Qin Li of Shenyang Agricultural University in north China.
Viral DNA preparation and sequencing
The procedures for isolating OBs and preparing viral DNA were as described by Cheng et al., 2005 . In brief, the OBs were purified by density gradient centrifugation, and were dissociated with a lysis buffer containing 0.1 mol/L Na2CO3 and 0.15 mol/L NaCl on ice for 30 min. After that, 0.5% SDS and proteinase K (50 mg/mL) were added and incubated at 37°C for 4 h. The digested solution was progressively extracted with phenol and chloroform mixtures. DNA was precipitated with 70% ethanol. The DNA was dried and dissolved in 2.0 mmol/L Tris (pH 8.0). The quantity and quality of the isolated DNA were determined spectrophotometrically and by electrophoresis on 0.7% agarose gel. A DNA fragment library of PhcyNPV was constructed using the shotgun method described by Zhu et al. . All clones were sequenced and the full-length sequence was constructed by the Chinese National Human Genome Center at Beijing.
ORFs in the PhcyNPV genome were identified using ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). All BLAST searches were done through the National Center for Biotechnology Information (NCBI) websites. The signal peptide data were downloaded from http://www.cbs.dtu.dk/services/SignalP/ using the software Prodotar v.1.03.
This work was supported by a grant from the China Agriculture Research System (Sericulture industry, CARS-22-ZJ0101).
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