Isolation and identification of a novel WSSV nucleocapsid protein by cDNA phage display using an scFv antibody

In a previous study, a scFv phage display library against white spot syndrome virus (WSSV) was constructed and yielded a clone designated A I with conformational specificity against native but not denatured viral antigen. Although the clone A1 has been used successfully as a diagnostic antibody, its precise target antigen has not been elucidated. A different strategy was adopted involving the construction of a second T7 phage display library utilizing mRNA isolated from shrimp infected with WSSV. Following RT-PCR and T7 phage library construction, phages displaying the candidate epitope were selected with A I scFv. Since successive enrichment steps were not associated with an increased titer of the phages, enrichment after successive tests was confirmed by PCR resulting in the prefer-red selection of a specific DNA sequence encoding a novel nucleocapsid protein WSSV388. Immune electron microscopy revealed that WSSV388 is located on the nucleocapsid. This result demonstrated that unknown antigen could be identified by phage display using the epitope conformation dependent scFv. (c) 2006 Elsevier B.V. All rights reserved.

Functional analysis of FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

The fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8-GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8-GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8-GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

Characterization and phylogenetic analysis of the chitinase gene from the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

A putative chitinase gene was identified within the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV, also called HaSNPIV) genome. The open reading frame (ORF) contains 1713 nucleotides (nt) and encodes a protein of 570 amino acids (aa) with a predicted molecular weight of 63.6 kDa. Transcription started at about 18 h post infection (p.i.) and the protein was first detected at 20 h p.i. The times of transcription and expression are characteristic of a late baculovirus gene. 5' and 3' RACE indicated that transcription was initiated from the adenine residue located at -246 nt upstream from the ATG start site and the poly (A) tail was added at 267 nt downstream from the stop codon. This is the first report on the molecular characterization of a chitinase from a single nucleocapsid NPV. The phylogeny of baculoviral chitinase genes were extensively examined in comparison with chitinases derived from bacteria, fungi, nematode, actinomycetes, viruses, insects and mammals. Neighbor-joining and most parsimony analyses showed that the baculoviral chitinases were clustered exclusively within gamma-proteobacteria. Our results strongly suggest that baculoviruses acquired their chitinase genes from bacteria. (C) 2004 Elsevier B.V. All rights reserved.

Cloning of biologically active genomes from a Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus isolate by using a bacterial artificial chromosome

Purification of genotypes from baculovirus isolates provides understanding of the diversity of baculoviruses and may lead to the development of better pesticides. Here, we report the cloning of different genotypes from an isolate of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) by using a bacterial artificial chromosome (BAC). A transfer vector (pHZB10) was constructed which contained an Escherichia coli mini-F replicon cassette within the upstream and downstream arms of HaSNPV polyhedrin gene. Hz2e5 cells were co-transfected with wild-type HaSNPV DNA and pHZB10 to generate recombinant viruses by homologous recombination. The DNA of budded viruses (BVs) was used to transform E. coli. One of the bacmid colonies, HaBacHZ8, has restriction enzyme digestion profiles similar to an in vivo cloned strain HaSNPV-G4, the genome of which has been completely sequenced. For testing the oral infectivity, the polyhedrin gene of HaSNPV was reintroduced into HaBacHZ8 to generate the recombinant bacmid HaBacDF6. The results of one-step growth curves, electron microscopic examination, protein expression analysis and bioassays indicated that HaBacDF6 replicated as well as HaSNPV-G4 in vitro and in vivo. The biologically functional HaSNPV bacmids obtained in this research will facilitate future studies on the function genomics and genetic modification of HaSNPV. (C) 2003 Elsevier B.V. All rights reserved.

HIV-1核衣壳蛋白NCp7的功能及其抑制剂研究进展

NCp7在HIV-1生活周期中多个步骤发挥重要的核酸分子伴侣作用.抑制NCp7的功能可阻断HIV-1复制.NCp7含有两个高度保守的锌指结构,对NCp7的功能起决定性作用.锌指结构突变将导致HIV-1丧失感染能力.NCp7抑制剂可望成为下一代不容易产生耐药突变的抗HIV-1药物.本文就NCp7抑制剂目前的研究进展作简略综述.

Purification and characterization of White Spot Syndrome Virus (WSSV) produced in an alternate host: crayfish, Cambarus clarkii

Penaeid shrimp is the natural host of White Spot Syndrome Virus (WSSV) that can cause high mortality in the infected hosts. Attempts to obtain sufficient amounts of purified intact WSSV for characterization have been unsuccessful. Using crayfish, Cambarus clarkii as a proliferation system, a large amount of infectious WSSV was reproduced and intact WSSV viral particles were purified with a new isolation medium by ultra-centrifugation. Purified WSSV particles were very sensitive to organic solvents and the detergent, Triton X-100. The size of the rod-shape, somewhat elliptical, intact WSSV was 110-130 x 260-350 mm with a long, tail-like envelope extension. The naked viral nucleocapsid was about 80 x 350 nm, and it possessed 15 spiral and cylindrical helices composed of 14 globular capsomers along its long axis, and a 'ring' structure at one terminus. Distinct WSSV genome DNA patterns were obtained when the purified genomic dsDNA of WSSV was digested with five different restriction enzymes (HindIII, XhoI, B(BamHI, SalI, and SacI). In addition, at least 13 major and distinct protein bands could be observed when purified intact WSSV viruses were separated by SDS-PAGE followed by Coomassie Brilliant R-250 staining. The estimated molecular weights of these proteins were 190, 84, 75, 69, 68, 58, 52, 44, 28, 27.5, 23, 19, and 16 kD, respectively. Both the 44 and 190 kD proteins were easily removed if the hemolymph from the: WSSV infected crayfish was transiently treated with 1%, Triton X-100 before it was subjected to gradient centrifugation, indicating that both of them are located on the surface of the viral envelope. These characteristics are consistent with WSSV isolated from the penaeid shrimp. (C) 2001 Elsevier Science B.V. All rights reserved.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100 nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found. (c) 2007 Elsevier Inc. All rights reserved.