Purification and some particle properties of anthriscus yellows virus, a phloem-limited semi-persistent aphid-borne virus

Anthriscus yellows virus (AYV), a phloem-limited virus transmitted in the semi-persistent manner by the aphid Cavariella aegopodii, was purified by treatment of leaf extracts with cellulasc, followed by differential and sucrose density gradient centrifugation. ‘The preparations contained isometric particles c. 29 nm in diameter which were unstable unless stored in buffer at pH 8.0 containing 1 mM CaCl₂,. The particles sedimented as two components, ’full‘ nucleoprotein particles with A₂₆₀/A₂₈₀= 1.83 containing about 42% nucleic acid, and ’empty‘ protein shells with A₂₆₀,/A₂₈₀= 0.73; their buoyant densities in CsCl solutions were 1.52 and 1.27 g/cm³. Respectively. Yields of ihe nircleoprotein particles were c. 1.75 mg/kg leaf tissue. The particles contained a single species of RNA, of mol. wt 3.6 × 10 “(10 000 nucleotides). Particle protein preparations contained four electrophoretic species, of mol. wt (× 10³) 35.0, 28.3, 23.3 and 22.3.C. aegopodii did not transmit AYV from purified preparations. A rabbit injected with AYV preparations produced antibodies that coated AYV particles in electron microscope tests, but gave variable reactions in immunosorbent electron microscopy (ISEM), depending on the composition of the medium. No reactions were obtained in enzyme-linked inimunosorbent asjay (ELISA). No serological relationship was detected in ISEM between AYV and any of 10 viruses that resembled it in one or more properties.     

A Safe Foot-and-Mouth Disease Vaccine Platform with Two Negative Markers for Differentiating Infected from Vaccinated Animals

Vaccination of domestic animals with chemically inactivated foot-and-mouth disease virus (FMDV) is widely practiced to control FMD. Currently, FMD vaccine manufacturing requires the growth of large volumes of virulent FMDV in biocontainment-level facilities. Here, two marker FMDV vaccine candidates (A₂₄LL3D_YR and A₂₄LL3B_PVKV3D_YR) featuring the deletion of the leader coding region (L^pro) and one of the 3B proteins were constructed and evaluated. These vaccine candidates also contain either one or two sets of mutations to create negative antigenic markers in the 3D polymerase (3D^pol) and 3B nonstructural proteins. Two mutations in 3D^pol, H₂₇Y and N₃₁R, as well as RQKP_9-12→PVKV substitutions, in 3B₂ abolish reactivity with monoclonal antibodies targeting the respective sequences in 3D^pol and 3B. Infectious cDNA clones encoding the marker viruses also contain unique restriction endonuclease sites flanking the capsid-coding region that allow for easy derivation of custom designed vaccine candidates. In contrast to the parental A₂₄WT virus, single A₂₄LL3D_YR and double A₂₄LL3B_PVKV3D_YR mutant viruses were markedly attenuated upon inoculation of cattle using the natural aerosol or direct tongue inoculation. Likewise, pigs inoculated with live A₂₄LL3D_YR virus in the heel bulbs showed no clinical signs of disease, no fever, and no FMD transmission to in-contact animals. Immunization of cattle with chemically inactivated A₂₄LL3D_YR and A₂₄LL3B_PVKV3D_YR vaccines provided 100% protection from challenge with parental wild-type virus. These attenuated, antigenically marked viruses provide a safe alternative to virulent strains for FMD vaccine manufacturing. In addition, a competitive enzyme-linked immunosorbent assay targeted to the negative markers provides a suitable companion test for differentiating infected from vaccinated animals.     

Beet curly top virus from phloem exudate of Amsinckia douglasiana

Amsinckia douglasiana when infected with beet curly top virus (BCTV) produces more exudate, which is highly infective, than any other known host. Attempts were made to purify BCTV from the phloem exudate of infected Amsinckia douglasiana by differential centrifugation and sucrose density gradient fractionation. A_260/280 ratio of virus preparations was 1.58; S values were 74 and 147. Infectivity was distributed among several fractions during density gradient purification. Electron microscopy revealed the presence of pentagonal and hexagonal particles of 14–23 nm, some of which were paired. The virus was insensitive to DNAse, RNAse, and trypsin when these enzymes were tested individually but infectivity was substantially decreased when the virus was subjected to trypsin treatment followed by DNAse, suggesting that DNA is associated with the beet curly top virus.     

Purification and properties of an oxygen-evolving Photosystem II reaction-center complex from spinach

A chlorophyll-protein complex, capable of photochemical water oxidation and consisting of only one extrinsic protein of 33 kDa in addition to six intrinsic proteins of the Photosystem II reaction center, has been isolated from spinach thylakoids by digitonin extraction, performed at pH 6.0, followed by chromatographic separations using DEAE-Toyopearl 650S as described briefly (Tang, X.S. and Satoh, K. (1985) FEBS Lett. 179, 60–64). The protein complex contained approx. 3–4 manganese atoms, 2 mol plastoquinone-9 and 2 mol low-potential forms of cytochrome b-559 heme per mol of the photoactive primary acceptor, Q_A. The oxygen evolution of the complex was highly stimulated by the presence of CaCl₂ and stabilized by glycerol; the typical rate of 400–500 μmol O₂ per mg Chl per h was attained with 2,5-dichlorobenzoquinone and potassium ferricyanide as electron acceptors in the presence of 50 mM CaCl₂. The protein complex exhibited a dark-stable EPR Signal II; the microwave power saturation profile of the signal was almost identical with that of oxygen-evolving membrane preparations. The multiline EPR signal ascribable to Kok's S₂-state was elicited in this protein complex by illumination at 200 K, as in membrane preparations. These results indicate that the basic machinery of photosynthetic water oxidation is preserved in an almost intact state in the isolated chlorophyll-protein complex.     

Isolation and characterization of six pathogenesis-related (PR) proteins of Samsun NN tobacco

The purification to homogeneity of pathogenesis-related (PR) proteins R and S from Nicotiana tabacum cv. Samsun NN leaves has been achieved by using a combination of conventional and high-performance chromatographic supports. The same procedure allowed the purification and the characterization of four other proteins which displayed some properties characteristic of tobacco PR proteins and were shown to accumulate in tobacco leaves in response to virus infection. They can be, therefore, considered as new tobacco PR proteins which we designate as PR-s₁,-s₂,-r₁ and-r₂. The relative electrophoretic mobilities (Rf) under non-denaturing conditions were estimated to 0.30 for PR-r₁ and-r₂, 0.25 for Pr-R, 0.20 for PR-s₁ and-s₂ and 0.15 for PR-S. On SDS gels PR proteins R and S possessed the same apparent molecular weight (M _r 24000) as did PR-proteins s₁ and r₁ (M _r 14500) and PR-s₂ and-r₂ (M _r 13000). However, proteins s₁, s₂, r₁ and r₂ had identical electrophoretic mobilities on SDS gels when the loading sample buffer contained no reducing agent. Polyclonal antisera were raised against PR proteins R and S and used in immunoblotting experiments. Proteins R and S were shown to be serologically closely related. No cross-reaction was detected with any of the four new tobacco PR proteins r₁, r₂, s₁ and s₂ or with the previously described PR proteins, i.e. PR-1a,-1b,-1c,-2,-N,-O,-P and-Q.     

Properties of a carlavirus causing a latent infection of pepino (Solanum muricatum)

Pepino (Solanum muricatum) cuttings imported from Chile contained a latent virus which was transmitted by inoculation of sap to Chenopodium quinoa but not to 21 other species. The virus was transmitted by the aphid, Myzus persicae. In C. quinoa sap, the virus lost infectivity when diluted between 10^-3 and 10^-4, heated for 10 min between 65 and 70 °C, or stored at room temperature for 4 to 6 days. The virus particles were straight or slightly flexuous filaments 660 to 680 nm long. Up to 15 mg virus per 100 g C. quinoa leaves was obtained by clarification with a mixture of chloroform and carbon tetrachloride. Purified preparations had A_max/A_min= 1.11, A₂₆₀/A₂₈₀= 1–30, A^0.₂₆₀^1%= 2.8, and contained a single sedimenting component with a sedimentation coeficient of 149s and a buoyant density in CsCl of 1–318. The virus particles contained 5.5% of single-stranded RNA of mol. wt 2.4×10⁶ (estimated by gel electrophoresis of undenatured RNA) and sedimentation coefficient 38.5S, and a single polypeptide of mol. wt 33 000. The virus is distantly serologically related to potato S and carnation latent viruses and is considered a new member of the carlavirus group. The name pepino latent virus is proposed. The cryptogram for this virus is R/1: 2.4/5–5: E/E: S/Ve/Ap.     

On the Mechanism of Inactivation of Chikungunya Virus by Tannic Acid

The mechanism of the action of tannic acid (TA) on Chikungunya virus (CHIKV) was investigated. Both infectivity and hemagglutination (HA) activity of CHIKV were reduced by treatment with TA in vitro. Aggregation of the TA-treated virus particles was observed by electron microscopy. The reaction was reversible, depending on the pH of the mixture. However, mere dilution of the TA-virus mixture or addition of other protein, such as bovine serum albumin, did not restore the lost infectivity. TA also suppressed the infectivity of RNA extracted from the virus and the HA activity of the viral membrane. The affinity between TA and CHIKV structural proteins (E₁, E₂, and C) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a discontinuous buffer system and the order of affinity was found to be C>E₁>E₂. Specific conditions for binding of TA to each of the virus proteins were investigated.     

Cellulose acetate electrophoresis of soluble lens proteins of different species of mammals; the use of discontinuous buffer systems

• 1.1. Cellulose acetate electrophoresis using a Tris-veronal discontinuous buffer system, has been performed with soluble lens proteins of different species of mammals and compared with two conventional continuous buffers.
• 2.2. In the four species investigated (mouse, Mus musculus; rat, Rattus norvegicus; bull, Bos taurus; ovine, Ovis aries) a higher migration rate and an improved fractionation were obtained with the discontinuous buffer.
• 3.3. The advantages and application to the lens protein research is indicated.

     

High Resolution Polyacrylamide Gradient Gel Electrophoresis

A method was developed for high resolution electrophoresis of proteins in linear gradient (3 to 30%) polyacrylamide gel rods in a neutral phosphate buffer containing 0.1% sodium dodecyl sulfate. Well-defined protein zones were observed and improved resolution was attained especially for low molecular weight proteins in preparations containing a variety of polypeptides, e. g. viruses that are often separated by continuous gel methods. Electropherograms of continuous (8%)and gradient (3 to 30%) gels were made of purified vesicular stomatitis virus, variola virus, Rickettsia rickett-sii, and alpha and beta chains of hemoglobin in order to demonstrate the resolution of the gradient system.     

Arracacha virus B, a second isometric virus infecting arracacha (Arracacia xanthorrhiza; Umbelliferae) in the Peruvian Andes

Arracacha virus B (AVB), a previously undescribed virus, was found together with arracacha virus A or with a 750 nm flexous filamentous virus in arracacha (Arracacia Xanthorrhiza; Umbelliferae) growing in the Huanuco region of the Peruvian Andes. AVB was transmitted by inoculation of sap to 30 species from eight families out of 45 species from 10 families tested. It was transmitted through seed of Chenopodium quinoa but not by Myzus persicae. AVB was best propagated in C. Quinoa or Tetragonia expansa and assayed in C. quinoa, C. murale or C. amaranticolor. Sap from infeted <C. Quinoa was occasionally infective after dilution to 10^-4 but not 10^-5, after 10 min at 65 d? C but not 70 d? C, and after 12 but not 14 days at 20 d? C. In neutral phosphotungstate, AVB has isometric partilces c. 26 nm in diameter with a hexagonal profile. About 50- 150 A1 cm₂₆₀ units of purified virus were obtained from 1 kg infected C. quinoa leaf by extraction in 0.5 M phosphate buffer at pH 7.5, containing 0.05 M ethylene-daiminetetra-acetate (EDTA) and 0.2% mercaptoethanol, and clarificatin with chloroform, followed by two precipitations with polyethylene glycol and three cylces of differential centrifugation. Purified virus coefficent (Sd?_{20 w},) of 126 S and A₂₆₀/A₂₈₀ ratio of 1.80, bnut formed two isopycnic bands in CsC1 of buoyant density 1.481 and 1.492 g/cm³ with estimated nucleic acid contents of 40 and 41% respectively. AVB particles contained two proteins of mol.wt 26 000 (major component) and 20 000. AVB was not serologically related to any of 20 other morphologically similar viruses. Its properties suggest that it does not fall into any recognised group of viruses. the cryptogram of AVB is */*:*/40–41:S/S:S/*     

Determination of the specific radloactivity of proteins separated by two-dimensional gel electrophoresis

Tritlum-labeled proteins, separated by two-dimensional gel electrophoresis, can be quantitatively extracted using sodium dodecyl sulfate (SDS)-urea buffer and subsequently acid-precipitated in the presence of serum albumin as carrier at low temperature (0–4°C). Their radioactivity can be counted efficiently under these conditions. Besides a better efficiency of counting, this method has some other advantages over the classical procedures using H₂O₂. The amounts of the eluted proteins can be easily measured in the SDS solution, using the Lowry method, and therefore specific radioactivity can be calculated. Also SDS can be removed easily, and the proteins can be used for further experiments.     

Soybean 11S globulin polypeptides have an antigenic homology with 11S globulins from various plants

Summary Rabbit serum antibodies (AB) against glycinin acidic polypeptides were separated by cross exhausting, and the antibody fractions for each of the two subfamilies of glycinin subunits (A₁ and A₃) were obtained. The antibodies were used in the immuno blot assay with seed protein of various plant classes. Polypeptides homologous to soybean glycinin were detected. Homology with A₁ polypeptide was revealed in more cases than with A₃. Total seed protein preparations were subjected to centrifugation in sucrose density gradient, and the polypeptides, imunochemically related to glycinin, occurred only in fractions with sedimentation constant about 11S. The nativity of conservative antigenic determinants of 11S globulins is discussed.     

Comparison of Three Plant Rhabdovirus Isolates by Two Different Serological Techniques

Three different rhabdovirus isolates, eggplant mottled dwarf virus (EMDV), tomato veinyellowing virus (TVYV) and a hitherto undefined isolate from tomato in Portugal (Tom-P) were compared by two different serological techniques, immunosorbent electron microscopy (ISEM) and electro-blot-immunoassay (EBIA). Antisera were prepared either against purified virus or against partially purified virus preparations extracted by a one-step procedure. Even the partially purified virus preparations yielded antisera that allowed unequivocal identification of two of the five structural virus proteins, G and N, in EBIA and were suitable for specific trapping of complete virus particles on electron microscope grids. With both serological techniques identical results were obtained indicating a close serological relationship between the three isolates tested. Cross-reactions between the G proteins could be deduced from heterologous trapping of complete virus particles in ISEM. The cross-reactions for the G proteins were substantiated by the EBIA-results which indicated in addition cross-reactions between the remaining three major structural virus proteins, N, M₁, and M₂, but also revealed significant differences in the molecular weights of the G and M₁ proteins between TVYV and the other two isolates. The results obtained indicate that Tom-P is serologically closely related and in respect of protein molecular weights identical to EMDV, and that TVYV is not a separate virus but rather a strain of EMDV.     

Analysis of low molecular weight plasma proteins using ultrafiltration and large gel two‐dimensional electrophoresis

In this study, various solvent systems were applied to obtain a high and consistent recovery rate of low molecular weight plasma proteins (LMPP) from human plasma. A buffer system containing 7 M urea, 2 M thiourea, 25 mM NH₄HCO₃ + 20% ACN (pH 8.2) produced the highest recovery rate of LMPP. To validate the recovery of cut off membrane (COM) obtained using the urea buffer system, 27 different 30 kDa COMs were used to prepare the LMPP sample which were then subjected to 1‐D SDS‐PAGE. Statistical analysis showed that the buffer system with COM produced a consistent the recovery of LMPP. In addition, 2‐DE analysis was also conducted to determine the relative intensity of each protein spot. When molecular weight ranges over 30 kDa and under 30 kDa were evaluated, 953 and 587 protein spots were observed in the gels, respectively, resulting in a total of 1540 protein spots being resolved. Identification of the major proteins were then performed using a nano‐LC/MS system comprised of an HPLC system and an ESI‐quadrupole IT MS equipped with a nano‐ESI source.     

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.     

An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly‐γ‐glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self‐assembles as mono‐disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)‐solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET_A) in their active conformations. Furthermore, ET_A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.     

口蹄疫O型重组病毒的构建及其免疫原性分析

【目的】利用反向遗传操作技术,构建含O型口蹄疫病毒(food-and-mouth disease virus, FMDV) 3个拓扑型免疫优势结构蛋白基因的重组FMDV,评估其作为猪O型口蹄疫(food-and-mouth disease, FMD)疫苗候选株的潜力。【方法】通过基因合成,在FMD疫苗株O/HN/CHA/93 (古典中国拓扑型)的基因中嵌合流行株O/NXYCh/CHA/2018 (东南亚拓扑型) VP1结构蛋白的重组病毒骨架上,用O/TUR/5/2009疫苗株(中东-南亚拓扑型) VP1蛋白的G-H环基因替换其对等基因,构建含O型3个拓扑型FMDV结构蛋白基因的重组全长质粒,Not I线性化后转染表达T7 RNA聚合酶的BSR/T7细胞,拯救重组病毒。通过RT-PCR、序列测定、间接免疫荧光鉴定重组病毒;噬斑试验和一步生长曲线分析重组病毒的生物学特性。重组病毒制备疫苗免疫猪,用病毒中和试验分析其对当前流行的O型3个拓扑型FMDV的交叉反应性。【结果】成功拯救到含O型3个拓扑型FMDV结构蛋白基因的重组病毒,重组病毒与亲本病毒具有相似的生物学特性。亲本病毒和重组病毒制备的疫苗免疫猪,均能够对中东-南亚型(Middle East-South Asia, ME-SA)拓扑型和东南亚型(South-East Asia, SEA)拓扑型病毒株产生保护性平均中和抗体(>1.65log₁₀);均不能对古典中国型(Cathay)拓扑型流行株产生保护性平均中和抗体(<1.65log₁₀),但与亲本病毒相比,O/TUR/5/2009疫苗株G-H环基因的替换显著提高了对ME-SA和SEA拓扑型病毒株的交叉反应性(p<0.05)。【结论】本研究对未来FMD疫苗的设计具有重要的指导意义。     

Maturation Defects in Temperature-sensitive Mutants of Sindbis Virus

Temperature-sensitive mutants of Sindbis virus, which synthesize viral ribonucleic acid (RNA) but not mature virus at the nonpermissible temperature, were selected for the study of viral maturation. Of these, three mutants which complement each other genetically were used. Two major proteins, the nucleocapsid and membrane proteins, located, respectively, in the viral nucleoid and membrane, were found in intact virions. In cells infected with wild-type Sindbis virus, four distinct types of viral RNA with sedimentation coefficients of 40S, 26S, 20S, and 15S were detected in constant distribution. The 20S RNA was ribonuclease-resistant, whereas the other types were ribonuclease-sensitive. The 40S RNA, identical to that obtained from the virion, was found associated with nucleocapsid protein as a subviral particle, which was assumed to be the nucleoid. Viral materials from cells infected with the mutants under nonpermissive conditions were compared with those from cells infected with wild-type virus, in terms of (i) the distribution of the different types of RNA, (ii) the association of infectious viral RNA into subviral particles, and (iii) the ability of infected cells to hemadsorb goose erythrocytes. According to these criteria, each of the three mutants demonstrated different maturation defects. Defective nucleocapsid proteins and membrane proteins may each account for one of the above mutants. The thrid mutant may have defects in a minor structural protein or possibly a maturation protein which is involved in the assembly of Sindbis virus.     

Particle properties of parsnip yellow fleck virus

Particle preparations of parsnip yellow fleck virus (PYFV) isolates A-421 and P-121, representing the two major serotypes, were made by clarifying leal extracts with ether or butan-1-ol and concentrating the virus particles by precipitation with polyethylene glycol and differential centrifugation. The preparations contained c. 31 nm-diameter particles comprising two sedimenting components. Top component (T) consisted of stain-penetrable protein shells with A₂₆₀/A₂₈₀= 0.8–0.9, sedimentation coefficient (S₂₀) = 56 S (A-421) or 60 S (P-121), and buoyant density = 1.297 g/cm³. Bottom component (B) consisted of nucleoprotein particles, not penetrable by negative stain, with A₂₆₀/A₂₈₀= 1.9, sedimentation coefficient (S⁰_20.w) = 148 S (A-421) or 153 S (P-121), and buoyant density = 1.520 g/cm³ (A-421) or 1.490 g/cm³ (P-121). Yields of B component particles were up to c. 1 mg/100 g leaf tissue (both isolates); yields of T component particles were up to c. 0.6 mg (A-421) or 5.5 mg (P-121) per 100 g leaf tissue. PYFV particles were found to contain a single RNA species (mol. wt c. 3.4 × 10⁶, c. 9800 nucleotides), constituting 40% of the particle weight, and three polypeptide species, of mol. wt (× 10 ³) 30 , 26 and 24 (A-421) or 31 , 26 and 23 (P-121).