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

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.     

Plasmids captured in <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">metallidurans</Emphasis> CH34: defining the PromA family of broad-host-range plasmids

The self-transmissible, broad-host-range (BHR) plasmid pMOL98 was previously isolated from polluted soil using a triparental plasmid capture approach and shown to possess a replicon similar to that of the BHR plasmids pSB102 and pIPO2. Here, complete sequence analysis and comparative genomics reveal that the 55.5 kb nucleotide sequence of pMOL98 shows extensive sequence similarity and synteny with the BHR plasmid family that now includes pIPO2, pSB102, pTER331, and pMRAD02. They share a plasmid backbone comprising replication, partitioning and conjugative transfer functions. Comparison of the variable accessory regions of these plasmids shows that the majority of natural transposons, as well as the mini-transposon used to mark the plasmids, are inserted in the parA locus. The transposon unique to pMOL98 appears to have inserted from the chromosome of the recipient strain used in the plasmid capture procedure. This demonstrates the necessity for careful screening of plasmids and host chromosomes to avoid mis-interpretation of plasmid genome content. The presence of very similar BHR plasmids with different accessory genes in geographically distinct locations suggests an important role in horizontal gene exchange and bacterial adaptation for this recently defined plasmid group, which we propose to name “PromA”. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of antiapoptosis genes on clarification performance

Optimal bioreactor harvest time is typically determined based on maximizing product titer without compromising product quality. We suggest that ease of downstream purification should also be considered during harvest. In this view, we studied the effect of antiapoptosis genes on downstream performance. Our hypothesis was that more robust cells would exhibit less cell lysis and thus generate lower levels of cell debris and host‐cell contaminants. We focused on the clarification unit operation, measuring postclarification turbidity and host‐cell protein (HCP) concentration as a function of bioreactor harvest time/cell viability. In order to mimic primary clarification using disk‐stack centrifugation, a scale‐down model consisting of a rotating disk (to simulate shear in the inlet feed zone of the centrifuge) and a swinging‐bucket lab centrifuge was used. Our data suggest that in the absence of shear during primary clarification (typical of depth filters), a 20–50% reduction in HCP levels and 50–65% lower postcentrifugation turbidity was observed for cells with antiapoptosis genes compared to control cells. However, on exposing the cells to shear levels typical in a disk‐stack centrifuge, the reduction in HCP was 10–15% while no difference in postcentrifugation turbidity was observed. The maximum benefit of antiapoptosis genes is, therefore, realized using clarification options that involve low shear, <1 × 10⁶ W/m³ and minimal damage to the cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:100–107, 2014     

Phenotypic transformation including host-range transition through superinfection of T-even phages

Mosaic genome design, considered evidence of horizontal gene transfer, is prominent in T-even phage tail fiber genes involved in host recognition. The possibility of direct gene transfer was assessed through superinfection with two virulent phages T2 and PP01, which caused host recognition shift. Two recombinant phages designated as TPr03 and TPr04 were isolated. PCR-restriction fragment length polymorphism analysis and sequence analysis suggested that 18% of the TPr03 and 38% of the TPr04 genome derived from PP01. Both isolates showed host ranges identical to PP01. The results suggested the possibility of generating various recombinant phages by intentional dual infections and of the occasional occurrence in nature of generation of phage showing new characteristics through superinfection, followed by the genomic recombination.     

Genetic basis of non-transposition of Tn5 in Pseudomonas aeruginosa following mobilisation of RP4 Mob::Tn5 from Escherichia coli

Abstract A Tn 5 transposon mutagenesis system based on mobilization of the narrow-host-range plasmid pACYC184 from Escherichia coli by a chromosomally integrated promiscuous plasmid RP4 was found to be non-applicable to Pseudomonas aeruginosa recipients. Transposition following mobilization was based on cloning an RP4 DNA fragment (/ RP4 Mob) into pACYC184 and Tn 5 transposition into the fragment (/ RP4 Mob::Tn5). It was shown by DNA sub-cloning of RP4 Mob::Tn 5 on to a wide-host-range plasmid vector that mobilization was unaffected but that reduced survival of the vector or host following mobilization was responsible. However, mutagenesis was achieved by the provision of cloned RP4 Mob DNA in the P. aeruginosa recipients.     

A型流感病毒非结构蛋白NS1与宿主相互作用的研究进展

作为A型流感病毒的非结构蛋白,NS1蛋白是流感病毒的一个重要的毒力因子,决定了病毒在宿主细胞内的破坏作用。概述了NS1蛋白对宿主细胞蛋白合成、宿主细胞凋亡的影响,及其拮抗干扰素的作用。     

Identification of sequence motifs involved in Dengue virus–host interactions

Dengue fever is a rapidly spreading mosquito-borne virus infection, which remains a serious global public health problem. As there is no specific treatment or commercial vaccine available for effective control of the disease, the attempts on developing novel control strategies are underway. Viruses utilize the surface receptor proteins of host to enter into the cells. Though various proteins were said to be receptors of Dengue virus (DENV) using Virus Overlay Protein Binding Assay, the precise interaction between DENV and host is not explored. Understanding the structural features of domain III envelope glycoprotein would help in developing efficient antiviral inhibitors. Therefore, an attempt was made to identify the sequence motifs present in domain III envelope glycoprotein of Dengue virus. Computational analysis revealed that the NGR motif is present in the domain III envelope glycoprotein of DENV-1 and DENV-3. Similarly, DENV-1, DENV-2 and DENV-4 were found to contain Yxxphi motif which is a tyrosine-based sorting signal responsible for the interaction with a mu subunit of adaptor protein complex. High-throughput virtual screening resulted in five compounds as lead molecules based on glide score, which ranges from ?4.664 to ?6.52 kcal/Mol. This computational prediction provides an additional tool for understanding the virus–host interactions and helps to identify potential targets in the host. Further, experimental evidence is warranted to confirm the virus–host interactions and also inhibitory activity of reported lead compounds.     

ChikvInt: a Chikungunya virus–host protein–protein interaction database

Chikungunya is a fast-mutating virus causing Chikungunya virus disease (ChikvD) with a significant load of disability-adjusted life years (DALY) around the world. The outbreak of this virus is significantly higher in the tropical countries. Several experiments have identified crucial viral–host protein–protein interactions (PPIs) between Chikungunya Virus (Chikv) and the human host. However, no standard database that catalogs this PPI information exists. Here we develop a Chikv-Human PPI database, ChikvInt, to facilitate understanding ChikvD disease pathogenesis and the progress of vaccine studies. ChikvInt consists of 109 interactions and is available at www.chikvint.com .     

Properties of a host range and coat protein variant of broad bean true mosaic comovirus from Vicia faba

Unlike other described isolates of broad bean true mosaic comovirus (BBTMV), a variant, code name SB, infected some non-leguminous plant species and, in N. benthamiana, induced systemic mottling and puckering of the leaves. However, like other described BBTMV isolates, purified SB particle preparations contained isometric particles c. 28 nm in diameter that sedimented as two nucleoprotein components with S_{20, w} values of 90S and 109S; some preparations occasionally contained a component of c. 50S. Virus particles contained two ssRNA species which, when denatured in glyoxal, had estimated M_T values of 2.1 × 10⁶ and 1.3 × 10⁶ and co-electrophoresed with cowpea mosaic virus RNA-1 and RNA-2 respectively. Isolate SB was serologically indistinguishable from British and German isolates of BBTMV. However, SB virus particles contained a major polypeptide (L) of M_r between c. 31 000 and up to three minor ones (S) or M_r between c. 20 000 and 24 000. This contrasts with protein preparations from other BBTMV isolates that typically contain only two polypeptides of M_r c. 37 000 (L) and 21 000 (S). Following isopycnic centrifugation in CsCl, SB particles purified from pea separated into two major components with densities of 1.39 and 1.44 g cm^-3 and a minor component of estimated density 1.43 g cm^-3. In Cs₂SO₄, virus preparations separated into three major components with densities of 1.30, 1.32 and 1.36 g cm^-3 and a minor one of density 1.27 g cm^-3. In CsCl isopycnic gradients, SB particles purified from TV. benthamiana separated into two components with densities of 1.38 and 1.43 g cm^-3. During immuno-electrophoresis in agarose gels, freshly prepared virus and preparations stored for up to 4 days at 4°C contained a single component that migrated rapidly to the anode, whereas similar preparations of an English isolate of BBTMV migrated as a single component that moved only slowly toward the anode but which, within 48 h, contained an additional component with a migration rate similar to that of isolate SB. Isolate SB is therefore a host range variant of BBTMV which, in comparison with previously described isolates of BBTMV, has an increased negative charge of its particles prior to any appreciable degradation of its S protein, and S protein that is degraded less rapidly. These features probably account for the anomalies observed in isopycnic centrifugation.     

一种新的遍在蛋白融合基因Uba256的克隆及表达

斜纹夜蛾核多角体病毒 (Spodopteralituramulticapsidnucleopolyhedrovirus ,SpltMNPV)的Uba2 5 6基因是已知昆虫病毒基因组中惟一编码遍在蛋白与GP37融合蛋白的基因。通过PCR方法扩增得到该基因及其N端的遍在蛋白编码区与C端的GP37编码区。将这 3个基因片段分别克隆至质粒pBV2 2 0与pQE30 ,构建得到重组质粒pB VUBCP、pQEUB与pQECP。pBVUBCP转化大肠杆菌DH5α ,经热激诱导表达了一条 38kD的蛋白带 ,证明Uba2 5 6表达的蛋白在大肠杆菌中没有发生剪切。pQEUB、pQECP转化大肠杆菌M15 ,经IPTG诱导 ,Western印迹分析结果表明遍在蛋白与GP37蛋白获得高效表达。以Ni2 NTA偶联抗体检测证明所表达的蛋白质均为融合蛋白质。纯化的融合蛋白质免疫新西兰大白兔可诱导产生特异抗体。ELISA和Western印迹检测结果显示 ,SpltMNPV遍在蛋白抗体及GP37抗体均与表达的融合蛋白质呈阳性反应 ,表明所表达的融合蛋白质仍保持原有蛋白质的免疫原性。以获得的抗体对感染SpltMNPV 72h的Sl zsu 1细胞进行检测 ,发现仅有一条 34kD的蛋白质带与GP37抗体发生特异反应 ;而有 4条分子量分别为 14、2 4、33、5 1kD的蛋白质带与遍在蛋白抗体发生特异反应 ,表明Uba2 5 6基因表达的蛋白质在宿主细胞内发生了剪切。