Cesium Chloride Density Research Articles

New insights of Sacbrood virus.

Over the last several years, major efforts have been expended to study viral infection of honeybees mainly due to colony losses around the world (Allen M, et al., 1996). It seems that honeybees are infected with numerous viruses mounting to 18 so far. Infection may be asymptomatic but could still have adverse effects on the bee and may even cause death resulting in colony collapse. Sacbrood virus (SBV) is the most widely distributed of all honey bee viruses. Since its first identification in Apis mellifera L in the United State in 1913, infection of SBV has been found in almost all colonies throughout the world (Allen M, et al., 1996; Ellis J D, et al., 2005; Kim Cuc N T, et al., 2008; Ma M X, et al., 2010). SBV infects the brood and adults, but 2-day old larvae appear to be the most susceptible resulting in death (Ritter W, 1996). Diseased larvae do not pupate and ecdysial fl uid collects around the integument in the form of a “sac”, after which the disease has been designated. The color of infected larvae changes from pearly white to pale yellow. Following death, larvae dry out, making a dark brown gondola-shaped scale (Bailey L, 1975). SBV may infect adults without overt signs of disease (Anderson D L, et al., 1989; Bailey L, 1969). However, life span may become shorter (Bailey L, 1969; Wang D I, et al., 1970). Sacbrood occurs most frequently in the spring when the colony is in a very active growing phase (Bailey L, 1969). The SBV was firstly described infecting the eastern honeybee, A. cerana, in Guangdong China in 1972 and was designated the Chinese sacbrood virus (CSBV). It emerged once again in 2008 in Liaoning, China (Ma M X, et al., 2010) causing death to individual bees or the collapse of the whole colony in an epidemic outbreaks. Later, the SBV was identified in A. cerana in Thailand and in Korea in 1982 and 2008, and named Thi SBV (TSBV) and AcSBV-Kor, respectively. SBV is 26-30 nm in diameter, icosahedral, pseudo T=3 structure symmetry, nonenveloped, and featureless in appearance with sedimentation coefficient of 160S and a buoyant density in cesium chloride of 1.33 g, and belongs to Picornaviridae. The outer shell of the capsid is composed of 60 repeated structures, each consisting of one molecule of 3 subunits VP1, VP2, and VP3 (25, 28, and 31.5 kDa), but each consisting of a single molecule of four subunits VP1, VP2, VP3, and VP4 (30.5, 31.5, 37.8 kDa and 44.2 kDa, respectively) about CSBV. The capsid proteins play important roles in the protection and in the determination of host specificity, tissue tropism and regional variations. Based on phylogeny and sequence homology of SBV-VP1, the SBV strains were divided into two major groups, an AC genotype infecting Apis cerana and an AM genotype infecting Apis mellifera, and also that the virus has host specificity and regional variations (Ma M X, et al., 2013).

RNA-Friendly Plasmid Preparation

With the advent of miniprep kits for the preparation of plasmids, we have noticed that many investigators use plasmids prepared by these methods as transcription templates or for cell transfection experiments. This can be quite problematic because miniprep (or maxiprep) DNA can often be contaminated with RNases. Indeed, most DNA preparation kits include pancreatic RNase. This RNase is extremely difficult to inactivate and can contaminate benchtops and pipettors. We highly recommend that plasmid DNAs, especially those that will be used repeatedly, be purified using traditional cesium chloride (CsCl) density gradient methods that do not involve the use of RNase. We have found that the protocol described here yields highly pure, noncontaminated plasmid DNA suitable for in vitro transcription and/or transfection analyses. An added benefit to this procedure is that it will yield enough plasmid DNA for many, many experiments.

Physical and Genomic Characteristics Identify Chicken Proventricular Necrosis Virus (R11/3 Virus) as a Novel Birnavirus

Chicken proventricular necrosis virus (CPNV), isolate R11/3, previously was isolated from transmissible viral proventriculitis-affected chickens and was determined to be the likely etiology of this disease. CPNV was identified as a birnavirus on the basis of virion size and morphology (icosahedral, approximately 75 nm in diameter, nonenveloped); buoyant density in cesium chloride (1.32 g/ml); a genome comprising bisegmented, double-stranded RNA (approximately 3.8 and 3.4 kilobase pairs); and nucleotide sequence analyses. Nucleotide sequencing of CPNV RNA, segment B, identified a single large open reading frame that encodes a 903-amino acid protein. The 903-amino acid protein was identified as the putative VP1, the viral RNA-dependent RNA polymerase (RdRp), on the basis of sequence homologies with other birnavirus VP1 proteins. The CPNV VP1 possessed the unique permuted RdRp sequence motif arrangement characteristic of birnaviruses; however, phylogenetic analyses based on VP1 demonstrated that CPNV is deeply divergent from other birnaviruses.

The conversion of tomato-fruit polygalacturonase isoenzyme 2 into isoenzyme 1 in vitro.

Polygalacturonase is extractable from ripe tomatoes in two isoenzyme forms, polygalacturonase 1 and 2. These isoenzymes have previously been shown to have substantially different properties although their polypeptides appear similar. Green fruit contain a heat-stable, non-dialysable factor capable of the conversion of polygalacturonase 2 in vitro into another isoenzyme which, on the basis of heat stability, molecular weight and density in caesium chloride, is equivalent to polygalacturonase 1. The amount of this factor extractable from tomato tissue increases during ripening.

A Novel Mite-Transmitted Virus with a Divided RNA Genome Closely Associated with Pigeonpea Sterility Mosaic Disease

ABSTRACT The agent of sterility mosaic, a disease that is a major constraint on pigeonpea (Cajanus cajan) production in the Indian subcontinent, is transmitted by the eriophyid mite, Aceria cajani. This agent has remained elusive for decades despite intensive efforts but we report the isolation of highly flexuous filamentous virus-like particles (VLPs) of 3 to 10 nm in width and of undefined lengths from sterility mosaic disease (SMD)-affected pigeonpea plants. Purified VLP preparations from virus-infected pigeonpea and Nicotiana benthamiana had a buoyant density in cesium chloride of 1.22 to 1.23 g cm(-3) and contained a major virus-specific protein species of approximately 32 kDa and 5 to 7 RNA species of approximately 6.8 to 1.1 kb. The sequence of some complementary DNA clones to RNA from purified VLP preparations had no significant matches in database searches. Two oligonucleotide primers derived from one such sequence, when used in reverse transcriptase-polymerase chain reaction assays, amplified a product of 321 bp specifically from SMD-affected pigeonpea plants. Purified VLP preparations were used to produce polyclonal antibodies that, in infected plants, detected the virus using enzyme-linked immuno-sorbent assay (ELISA) and the virus-specific 32-kDa protein in western immunoblotting (WIB). In such assays, the virus was detected consistently in all SMD-affected pigeonpea plant samples from several different locations in India, but not in samples from symptom-free pigeonpea plants from the same locations. In experimental studies, all pigeonpea plants inoculated with viruliferous A. cajani and those plants graft-inoculated with SMD-affected tissue were infected with the virus as assessed by ELISA and WIB, but not any uninfected pigeonpea plants. This virus, tentatively named Pigeonpea sterility mosaic virus (PPSMV), has some properties similar to virus species in the genera Tospovirus and Tenuivirus and with the eriophyid mite-transmitted High plains virus (HPV) but is distinct from these and from all other characterized viruses. The combination of novel properties shown by PPSMV and HPV suggest that they may constitute species in a new genus of plant viruses.

Purification of DNA for the transfection of a Spodoptera frugiperda cell line.

Spodoptera frugiperda (Sf-9) cells have been widely used in baculovirus expression systems, transient gene expression studies and transgenic cell lines. These applications commonly require the transfection of bacterial plasmid DNA. One of the most reliable methods of preparing transfection-quality plasmid DNA is cesium chloride (CsCl) density gradient centrifugation. However, the traditional CsCl DNA purification is a long and laborious process. We have made a series of modifications to the traditional method that makes it faster, safer and easier. In the current study we demonstrate that DNA prepared by our modified CsCl method was also better for the transfection of Sf-9 cells than DNA prepared by the traditional CsCl method.

Outbreak of gastroenteritis in military recruits associated with serotype 3 astrovirus infection.

A serotype 3 astrovirus was identified in stool samples from an outbreak of acute gastroenteritis that occurred among military recruits in France. Sixteen stools samples and eight pairs of acute- and convalescent-phase serum were collected from affected individuals. Astrovirus was detected in two stool samples by electron microscopy and in four stool samples by reverse transcriptase-polymerase chain reaction (RT-PCR). Seroconversion to the astrovirus present in one stool was detected in seven patients by using solid-phase immune electron microscopy (SPIEM) and dot blot. For three patients, the serological results were consistent with the PCR results, indicating that astrovirus was a cause of gastroenteritis in these young adults. This study describes the characterization of the serotype 3 astrovirus associated with this outbreak. The virus has a buoyant density in cesium chloride of 1.365 gm/ml and contains two proteins immuno-precipitated with rabbit serum. Only the larger protein was recognized by immunoblotting using a convalescent-phase human serum. The protein composition of this virus differs from that reported for serotype 1 astrovirus, indicating heterogeneity in the capsid composition among astrovirus serotypes.

Characterization of a Potexvirus InfectingHostaspp.

A previously undescribed potexvirus, named Hosta virus X (HVX), was found in 17 naturally infected hosta (Hosta spp.) cultivars from Minnesota, Indiana, Illinois, Iowa, and Michigan. HVX was readily transmitted mechanically but infected only Nicotiana benthamiana and Hosta spp., in which symptoms ranged from severe mosaic and leaf necrosis to latency. Particles of HVX averaged 530 nm in length, had a buoyant density in cesium chloride of 1.28 gm/cm 3 , and contained a single genomic species of ssRNA approximately 3 kb in size. The capsid protein of HVX had a molecular mass of approximately 27 kDa. In indirect enzyme immunoassays, HVX reacted with an antiserum to clover yellow mosaic virus (C1YMV), and less strongly with antiserum to hydrangea ringspot virus (HRSV), but not with antisera to any of 14 other potexviruses tested. However, in reciprocal tests, CIYMV reacted only very weakly with HVX antiserum. HVX did not infect any of three cultivars of pea that were susceptible to infection by CIYMV, and CIYMV did not infect any of three hosta cultivars susceptible to infection by HVX. Spread of HVX infection in hosta appears to occur by vegetative propagation and accidental mechanical transmission, and management of the disease can be achieved by virus indexing and cultural practices that minimize the risk of virus spread to susceptible cultivars.

Characterization of a Group D Rotavirus

A rotavirus isolated from the intestinal contents of commercial ring-neck pheasant chicks was determined to be a group D rotavirus based on its RNA electropherotype and its serologic relatedness to known turkey group A and D rotaviruses using immune electron microscopy and agar-gel immunodiffusion assays. Further characterization revealed a buoyant density in cesium chloride of 1.347 g/cm3 for double-shelled particles and 1.365 g/cm3 for single-shelled particles. The average size for the pheasant-origin group D rotavirus was determined to be 80 nm for double-shelled particles and 70 nm for single-shelled particles. In contrast, the average size for a turkey group A rotavirus isolate was determined to be 73 nm for double-shelled particles and 60 nm for single-shelled particles. There was no evidence of viral replication by the pheasant-origin group D rotavirus (either cytopathic effect or increase in numbers of viral particles) after six passages in MA104 cells. The hemagglutinating activity of the pheasant origin group D rotavirus was compared with that of a turkey origin group A rotavirus. Differences and similarities were noted depending upon the species of erythrocytes employed and the temperature of incubation.

Unique amino acid substitutions in the capsid proteins of foot-and-mouth disease virus from a persistent infection in cell culture

Maintenance of a persistent foot-and-mouth disease virus (FMDV) infection in BHK-21 cells involves a coevolution of cells and virus (J. C. de la Torre, E. Martínez-Salas, J. Díez, A. Villaverde, F. Gebauer, E. Rocha, M. Dávila, and E. Domingo, J. Virol. 62:2050-2058, 1988). The resident FMDV undergoes a number of phenotypic changes, including a gradual decrease in virion stability. Here we report the nucleotide sequence of the P1 genomic segment of the virus rescued after 100 passages of the carrier cells (R100). Only 5 of 15 mutations in P1 of R100 were silent. Nine amino acid substitutions were fixed on the viral capsid during persistence, and three of the variant amino acids are not represented in the corresponding position of any picornavirus sequenced to date. Cysteine at position 7 of VP3, that provides disulfide bridges at the FMDV fivefold axis, was substituted by valine, as determined by RNA, cDNA, and protein sequencing. The modified virus shows high buoyant density in cesium chloride and depicts the same sensitivity to photoinactivation by intercalating dyes as the parental FMDV C-S8c1. Amino acid substitutions fixed in VP1 resulted in altered antigenicity, as revealed by reactivity with monoclonal antibodies. In addition to defining at the molecular level the alterations the FMDV capsid underwent during persistence, the results show that positions which are highly invariant in an RNA genome may change when viral replication occurs in a modified environment.

Parietaria mottle virus, a possible new ilarvirus from Parietaria officinalis (Urticaceae)

A previously undescribed virus, probably a new member of the ilarvirus group, was isolated from Parietaria officinalis showing symptoms of yellow mosaic or mottling. This virus, for which the name parietaria mottle virus (ParMV) is proposed, differs in host range from other ilarviruses. ParMV was purified from Chenopodium quinoa by sap clarification with chloroform, and differential and sucrose density gradient centrifugation.Purified particles were quasi‐isometric to ovoid with diameters of about 24, 29 and 36 nm; no bacilliform particles were detected. Buoyant density in caesium chloride, determined on glutaraldehyde‐fixed virus, was 1.35 g/cm3.An antiserum to ParMV was obtained with a titre of 1:32 in agar gel double‐diffusion tests. ParMV did not react with eight other viruses or virus strains belonging to the ilarvirus group or to pelargonium zonate spot virus.Polyacrylamide gel electrophoresis indicated that ParMV contains a single major protein species of estimated molecular weight 24300 Da and four RNA species of estimated molecular weight 1 37, 1.19, 0.86 and 0.36 × 106 Da.

Characterization of a New Picorna-like Virus Isolated from Aphids

Summary A new picorna-like virus, provisionally named aphid lethal paralysis virus (ALPV), was isolated from the aphid Rhopalosiphum padi. The virus particles are isometric with a diameter of 26 nm, a sedimentation coefficient of 164S and a density in caesium chloride of 1.34 g/ml. Virions contain a 9.7 kb polyadenylated, ssRNA and four polypeptides: three major polypeptides of M r 34400, 32000 and 31200 and one minor polypeptide of M r 40800. ALPV is serologically unrelated to R. padi virus of aphids but is serologically related to cricket paralysis virus. The properties of this virus indicate that it should be classified in the family Picornaviridae.

Some properties of pea stem necrosis virus isolated fro pea in Japan.

Pea stem necrosis virus (PSNV) was readily purified from infected pea plants by a chloroformbutanol procedure with yields of about 200mg/kg leaf tissues. Purified virus preparations contained a single sedimenting component with a sedimentation coefficient of 118S, buoyant density in cesium chloride of 1.35g/cm3 and nucleic acid content of 17%. Polyacrylamide gel electrophoresis demonstrated single nucleic acid component and single coat protein with estimated molecular weights of 1.53×106 and 38, 000 daltons, respectively. Nucleic acid extracted from the virus preparations was identified as single-stranded RNA by the following properties: positive orcinol reaction; susceptibility to RNase A both in 1×SSC or 0.1×SSC; increase of about 21% in maximum absorption and shift both the absorption maximum and minimum to higher wave-lengths by 4 nm on treatment with formaldehyde (1.8%). Preparations of purified particles of PSNV reacted with homologous antiserum (gel diffusion titer, 1:1, 024) but not with any antisera to 8 different plant viruses with isometric particles including tomato bushy stunt, galinsoga mosaic, carnation mottle or melon necrotic spot viruses. However, physicochemical properties were similar to those of carnation mottle and allied viruses.

Further evidence that dioscorea latent virus is a potexvirus

SUMMARYDioscorea latent virus (DLV) was isolated from Dioscoreafloribunda but was not detected in any of 37 cvs of D. alata, D. bulbifera, D. esculenta or D. rotundata from eight countries. It was readily sap‐transmitted to 13 of 34 species from five of 12 families; it induced symptomless systemic infection in Nicotiana benthamiana and N. megalosiphon, but only symptomless local infection in other hosts. DLV was stable in vitro: sap from infected N. megalosiphon was still infective after 10 min at 75–80 oC but not 85 oC, after dilution to 10‐6 but not 10‐7, and after at least 12 months at 23 oC. DLV was not transmitted through seed, by contact or by the aphids Aphis gossypii and Myzus persicae.DLV had filamentous particles most of which measured c. 350–900 nm in purified preparations, with two modal lengths of 445 and 875 nm; the particles sedimented as two components with sedimentation coefficients of 111 S and 131 S and had a buoyant density in caesium chloride of 1–33 g cm‐3. The virus had a single nucleic acid species with a mol. wt of 2–3 ± 04 × 106 and usually produced two protein bands (mol. wts 24 900 and 23 100) in SDS‐PAGE, although virus preparations made in the presence of chloroform yielded only the larger polypeptide. DLV was serologically distantly related to commelina X and lily X viruses, but not to 11 other established or possible members of the potexvirus group.These results provide further evidence that DLV is a distinct but definitive potexvirus.

Studies on melon necrotic spot virus disease of cucumber and on the control of the fungus vector (Olpidium radicale)

SUMMARYMelon necrotic leaf spot virus (MNSV) caused a major outbreak of a leaf necrosis disease of hydroponically‐grown cucumber plants at Humberside in 1983. The virus had c. 33 nm diam. particles which reacted serologically with MNSV antiserum of Dutch or American origin. Virus particles, which contained a single polypeptide (mol. wt 45 × 103) and a presumed RNA species (mol. wt 1.5 × 106), had a sedimentation coefficient (s20.w) of 134 S and a buoyant density in caesium chloride of 1.35 g/cm3. The virus was mechanically transmissible, confined to species of Cucurbitaceae, transmitted by zoospores of Olpidium radicale and retained in the resting spores of the fungus. MNSV is thus both water‐borne and soil‐borne. O. radicale zoospores were killed in <5 min in suspensions containing 20 μg/ml of the surfactant Agral (alkyl phenol ethylene oxide). The disease did not reappear in 1984 when the cucumber crops were fed with nutrients containing 20μg/ml Agral.

Purification and partial characterisation of beet mild yellowing virus and its serological detection in plants and aphids

SUMMARYBeet mild yellowing virus (BMW) was reversibly precipitated at temperatures below about 5°C and this property was used as a final step in a purification procedure which yielded about 1 mg virus/kg tissue. Purified virus was infective and had an A200/A280 ratio of about 1–8. BMW particles were isometric with a diameter of 26 nm, sedimented at 116 S, had a buoyant density in caesium chloride of 1.42 g/cm3 and a coat protein mol. wt of 25 400.An antiserum to BMW had a titre in immunodiffusion tests of 1/256 and was used in immunodiffusion tests, immunospecific electron microscopy (ISEM) and enzyme‐linked immunosorbent assay to demonstrate a close serological relationship between BMW and beet western yellows virus. BMW was readily detected by ISEM in plants and also in aphid vectors after treatment of aphid extracts with a chloroform:butanol mixture.

Inactivated Poliovirus Vaccine and Test Development at Connaught Laboratories Ltd.

Trivalent inactivated poliovirus vaccine (lPV) (MK) was made, purified, and inactivated according to the amended Rijks Instituut (The Netherlands) protocol from primary monkey kidney tissue grown on microcarrier cultures. Losses of the type 2 component due to adsorption to the glass ampule occurred with the purified vaccine preparation. This problem was solved by changing the diluent, and the vaccine was submitted for evaluation in clinical trials at Johns Hopkins (Baltimore, Md.). Phase 2 of the development was to standardize production of IPV from MRC-5 (human diploid) cells on microcarriers and otherwise follow the Rijks Instituut's method. Results of experimental trivalent vaccine production and testing showed that the number of effective doses harvested from MRC-5 cell cultures compared favorably with vaccine derived from monkey kidney. The yields could be further increased with stearyl tyrosine as adjuvant. Large-scale production using 200-liter fermenters is in progress. Poliovirus particles of various densities in cesium chloride can be found in any IPV preparation and give rise to different immunogenic responses. As shown in this paper, some of these virus fractions produce a low primary humoral antibody response but appear to be important for memory induction.

Posttransfusion Hepatitis Type A

Hepatitis A virus (HAV) transmission through blood is a rare but potential cause of posttransfusion hepatitis. We can now document such a case supported by laboratory evidence of HAV in the donor blood. A 10-year-old girl manifested icteric hepatitis A 31 days after receiving a single unit of packed RBCs from a donor who subsequently experienced hepatitis A and died in hepatic failure. Hepatitis A virus antigen was detected in the donor's hepatocytes and in plasma obtained from the original donor unit. The density in cesium chloride of the HAV antigenic activity from the liver and plasma ranged from 1.33 to 1.37 g/cu cm, which is similar to that reported for infectious HAV particles. The implicated donor plasma had normal aminotransferase levels and was negative for antibody to HAV. Inoculation of this plasma into a chimpanzee resulted in the development of hepatitis A 23 days later based on the appearance of fecal HAV antigen, hepatitis, and IgM anti-HAV seroconversion. These data clearly document the presence of HAV in the donor sample that produced posttransfusion hepatitis A.

Hydrangea mosaic virus, a new ilarvirus from Hydrangea macrophylla (Saxifragaceae)

SUMMARYA previously unrecognised virus isolated from Hydrangea macrophylla with chlorotic mosaic leaf symptoms in West Sussex was named hydrangea mosaic virus (HydMV). HydMV was mechanically transmitted without difficulty to four of 16 species from three of five families, and was seed transmitted in Chenopodium quinoa, but was not aphid transmitted.Although relatively unstable in vitro, HydMV was purified by clarifying leaf extracts by emulsification with chloroform and acidification with citric acid, followed by differential centrifugation and sucrose density gradient centrifugation. Purified virus incompletely separated on sucrose density gradients into three components (T. M and B) with sedimentation coefficients (so20w) of 86, 97, and 105 S respectively, but all particles had buoyant densities in caesium chloride of 1.37 g/cm3. Virus contained a single polypeptide species (mol. wt 26.4 times 103), appeared quasiisometric to ovoid or elliptical, and measured c. 28 times 30 (T), 30 times 30 (M) or 30 times 32–38 nm (B). Single‐stranded RNA species or mol. wt 1–25, 1–08, 0–83, 0–36 and 0–27 (RNA‐1 to 5 respectively) were obtained from virus preparations but mixtures containing RNA‐1 to 3 plus either RNA‐4 + 5 or the coat protein, were infective.These properties suggest that HydMV has affinities with ilarviruses, but it showed no serological relationship to any of six ilarviruses or 42 other viruses.

Variations in the buoyant density of foot-and-mouth disease virus strains.

Some of the factors which effect the buoyant density in caesium chloride of foot-and-mouth disease virus were investigated. Under standard conditions, the buoyant densities of a range of vaccine virus strains, representing all seven serotypes, were found to be unrelated to their potential of being formulated into effective vaccines. In some virus strains, a small amount of naturally occurring high and/or low density components were observed and these components were examined in more detail in one virus strain.