Shanying Zhu

Genomic sequence, organization and characteristics of a new nucleopolyhedrovirus isolated from Clanis bilineata larva

BackgroundBaculoviruses are well known for their potential as biological agents for controlling agricultural and forest pests. They are also widely used as expression vectors in molecular cloning studies. The genome sequences of 48 baculoviruses are currently available in NCBI databases. As the number of sequenced viral genomes increases, it is important for the authors to present sufficiently detailed analyses and annotations to advance understanding of them. In this study, the complete genome of Clanis bilineata nucleopolyhedrovirus (ClbiNPV) has been sequenced and analyzed in order to understand this virus better.ResultsThe genome of ClbiNPV contains 135,454 base pairs (bp) with a G+C content of 37%, and 139 putative open reading frames (ORFs) of at least 150 nucleotides. One hundred and twenty-six of these ORFs have homologues with other baculovirus genes while the other 13 are unique to ClbiNPV. The 30 baculovirus core genes are all present in ClbiNPV. Phylogenetic analysis based on the combined pif-2 and lef-8 sequences places ClbiNPV in the Group II Alphabaculoviruses. This result is consistent with the absence of gp 64 from the ClbiNPV genome and the presence instead of a fusion protein gene, characteristic of Group II. Blast searches revealed that ClbiNPV encodes a photolyase-like gene sequence, which has a 1-bp deletion when compared with photolyases of other baculoviruses. This deletion disrupts the sequence into two small photolyase ORFs, designated Clbiphr-1 and Clbiphr-2, which correspond to the CPD-DNA photolyase and FAD-binding domains of photolyases, respectively.ConclusionClbiNPV belongs to the Group II Alphabaculoviruses and is most closely related to OrleNPV, LdMNPV, TnSNPV, EcobNPV and ChchNPV. It contains a variant DNA photolyase gene, which only exists in ChchNPV, TnSNPV and SpltGV among the baculoviruses.

Cloning and sequence analysis of theAntheraea pernyi nucleopolyhedrovirusgp64 gene

Frequent outbreaks of the purulence disease of Chinese oak silkworm are reported in Middle and Northeast China. The disease is produced by the pathogenAntheraea pernyi nucleopolyhedrovirus (AnpeNPV). To obtain molecular information of the virus, the polyhedra of AnpeNPV were purified and characterized. The genomic DNA of AnpeNPV was extracted and digested withHindIII. The genome size of AnpeNPV is estimated at 128 kb. Based on the analysis of DNA fragments digested withHindIII, 23 fragments were bigger than 564 bp. A genomic library was generated usingHindIII and the positive clones were sequenced and analysed. Thegp64 gene, encoding the baculovirus envelope protein GP64, was found in an insert. The nucleotide sequence analysis indicated that the AnpeNPVgp64 gene consists of a 1530 nucleotide open reading frame (ORF), encoding a protein of 509 amino acids. Of the eightgp64 homologues, the AnpeNPVgp64 ORF shared the most sequence similarity with thegp64 gene ofAnticarsia gemmatalis NPV, but notBombyx mori NPV. The upstream region of the AnpeNPVgp64 ORF encoded the conserved transcriptional elements for early and late stage of the viral infection cycle. These results indicated that AnpeNPV belongs to group I NPV and was far removed in molecular phylogeny from the BmNPV.

Analysis of the genomic sequence of Philosamia cynthia nucleopolyhedrin virus and comparison with Antheraea pernyi nucleopolyhedrin virus

BackgroundTwo 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.ResultsWe 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.ConclusionsMost 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.

Killing of Escherichia coli by Exogenous Hydroxyl Radicals

The effects and mechanisms were studied on killing of Escherichia coli, a Gram negative bacterium by exogenous hydroxyl radical. The results showed that, exogenous OH· was efficient on killing of E. coli cells. Over 99% of them was killed in 0.5 mg/L OH· solution. And exogenous OH· could damage biomolecules of E. coli. The concentrations and integrities of DNA, RNA and intracellular protein decreased with the increasing of the concentration of OH·, and the degree of decreasing of intracellular soluble protein concentration was higher than that of DNA or RNA. This indicated that protein is the major target of OH· among biomolecules in the cell. After treated by OH·, the leakage of electrolytes from cells increased. In 0.5 mg/L OH· solution, the value of relative leakage reached to 16.21%, which might be severe and fatal to E. coli cell. It was speculated that cell membrane is the initial target of exogenous hydroxyl radical.

Analysis of oral infection and helicase gene of the nucleopolyhedroviruses isolated from Philosamia cynthia ricini and Antheraea pernyi

Abstract Philosamia cynthia ricini is an important commercial silkworm in Asia. In this report, a nucleopolyhedrovirus isolated from P. cynthia ricini (PhcyNPV) larva was purified and compared with Antheraea pernyi nucleopolyhedrovirus (AnpeNPV), a pathogen of A. pernyi, another commercial silkworm in China. The two viruses had similar polyhedral morphology and shared high sequence homologue of viral fragments including the p143 gene. However, the restriction fragments, digested with SalI, XhoI, HindIII and PstI, respectively, were different. The cross-infectivity of the two viruses was also tested. AnpeNPV caused 57% mortality in larvae of P. cynthia rici, whereas PcrNPV did not kill larvae of A. pernyi. Results indicated that PhcyNPV and AnpeNPV had closed relatedness, and that PhcyNPV might be a variant of AnpeNPV.

Development of a novel T-vector supporting efficient green-white screening

T-vectors play an important role in cloning of polymerase chain reaction products. In the present study, a novel pUEG-T vector was developed using enhanced green fluorescent protein (EGFP) as an indicator. To improve EGFP-based green-white screening, the lipoprotein mutant promoter, a strong constitutive promoter, was utilized to control the expression of egfp gene. Two other efficient expression elements, the ColE1 replication origin of pUC18 and the expression cassette of pET-28a, were also integrated into pUEG-T vector. Expression analysis demonstrated the efficient accumulation of active EGFPs in Escherichia coli DH5α cells carrying the T-vector precursor pUEG. In T-A cloning using pUEG-T vector, white colonies containing foreign DNA and green colonies having no insertion could be handily distinguished under normal white light, without any chemical inducer or chromogenic substrate. Furthermore, no false positive was observed in any of the tested white colonies. This proves that pUEG-T is an inexpensive, convenient and efficient T-vector.

Expression of EGFP driven by BmNPV orf4 promoter, a novel immediate early promoter

Bombyx mori nucleopolyhedrovirus (BmNPV) orf4 has been shown to be expressed at very early stage of Bm-NPV infection cycle. In this study, using transient expression experiment, we demonstrated for the first time that orf4 promoter is an immediate early promoter, indicating that orf4 may play a role in the immediate-early stage of BmNPV infection. Moreover, with the recently developed Bac-to-Bac/BmNPV baculovirus expression system and a modified pFast-Bac1 whose polyhedrin promoter was replaced with orf4 promoter, a recombinant bacmid baculovirus expressing enhanced green fluorescent protein (EGFP) under the control of orf4 promoter in Bombyx mori (Bm) cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced easily with other promoters to direct the expression of foreign genes by using this novel system but also laid the foundation for the rescue experiment of orf4 deletion mutant.

Study on the Envelope Fusion Proteins of Baculovirus

GP64 homologies, which are found in Group I NPVs, and F proteins (Fa proteins), which are utilized in Group II NPVs, are required for virion entry into the cell. And Group I NPV also encodes homologue of Fa protein, and was named Fb protein. However, Fb protein dose not appear to mediate cell fusion. To further researching these important viral envelope proteins, the expression of GP64 and Fb proteins in Group I NPVs were studied by Real-time quantitative PCR. The results indicated that Fb is not the evolution redundancy gene but an important function gene. In addition, we transfected the fragments of CbNPV Fa and ie-1 into different insect cell lines, and found that CbNPV Fa and ie-1 could not express in BmN and T.ni High Five cells but could express in Sf-9 and Spli cells.