Purification and characterization of enterovirus 71 viral particles produced from vero cells grown in a serum-free microcarrier bioreactor system

Background

Enterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.

Principal Finding

In this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >10⁶ TCID₅₀/mL by 6 days post infection when a MOI of 10⁻⁵ was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).

Conclusion

These results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH₁ and CL domains was deleted by substitution of Cys with Ala (Fab_ΔSS). DSC measurements showed that the Tm values of Fab_ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab_ΔSS. The resulting Fab (mutSS Fab_ΔSS) had the mutations H:V177C and L:Q160C in Fab_ΔSS, confirming the formation of the disulfide bond between CH₁ and CL. The thermostability of mutSS Fab_ΔSS was approximately 5 °C higher than that of Fab_ΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab_ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.     

Immunological and Biochemical Characterization of Coxsackie Virus A16 Viral Particles

Background

Coxsackie virus A16 (CVA16) infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD). There are currently no vaccine or effective medical treatments available.

Principal Finding

In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10⁶ the tissue culture''s infectious dose (TCID₅₀) per mL within 7 days post-infection when a multiplicity of infection (MOI) of 10⁻⁵ was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24–28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35–38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176–190.

Conclusion

These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.     

Separation and characterization of some ribonucleic acids from imaginal discs of Drosophila melanogaster

Summary The RNA obtained from mass isolated imaginal discs of Drosophila melanogaster cultured in vitro in ³H-5-uridine has been studied. A separation procedure employing benzoylated, naphthoylated DEAE cellulose column chromatography was used to obtain several fractions of RNA, including one consisting primarily of ribosomal RNA, and three others relatively free of rRNA and transfer RNA. Other than its behavior in BND-cellulose chromatography, the RNA fractions have been examined with the following procedures: (1) sucrose gradient centrifugation, (2) heat labilities with apparent shifts in S-values, (3) changes in apparent secondary structure due to monovalent cation concentration, (4) Cs₂SO₄–CsCl equilibrium gradient centrifugation, (5) dimethyl sulfoxide gradient centrifugation, (6) melting out studies in different salt (Na⁺) concentrations, (7) limited digestion with different kinds of RNase, (8) kinetics of labeling, and (9) DNA-RNA hybridization. Marked differences were found among the RNA fractions using these techniques. The addition of -ecdysone to the cultured imaginal discs induces the production of quantitatively and perhaps qualitatively different RNA when compared with RNA obtained from discs cultured without the hormone. The possible secondary structures of the RNA fractions are also discussed.     

Blood Viscosity and the Expression of Inflammatory and Adhesion Markers in Homozygous Sickle Cell Disease Subjects with Chronic Leg Ulcers

Objective

To determine differences in TNF-α, IL-1β, IL-10, sICAM-1 concentrations, leg hypoxia and whole blood viscosity (WBV) at shear rates of 46 sec^-1 and 230 sec^-1 in persons with homozygous S sickle cell disease (SCD) with and without chronic leg ulceration and in AA genotype controls.

Design

& Methods: fifty-five age-matched participants were recruited into the study: 31 SS subjects without leg ulcers (SS_n), 24 SS subjects with leg ulcers (SS_u) and 18 AA controls. Haematological indices were measured using an AC.Tron Coulter Counter. Quantification of inflammatory, anti-inflammatory and adhesion molecules was performed by ELISA. Measurement of whole blood viscosity was done using a Wells Brookfield cone-plate viscometer. Quantification of microvascular tissue oxygenation was done by Visible Lightguide spectrophotometry.

Results

TNF-α and whole blood viscosity at 46 sec^-1 and 230 sec^-1 (1.75, 2.02 vs. 0.83, 1.26, p<0.05) were significantly greater in sickle cell disease subjects than in controls. There were no differences in plasma concentration of sICAM-1, IL-1β and IL-10 between SCD subjects and controls. IL-1β (median, IQR: 0.96, 1.7 vs. 0, 0.87; p<0.01) and sICAM-1 (226.5, 156.48 vs. 107.63, 121.5, p<0.005) were significantly greater in SS_u group compared with SS_n. However there were no differences in TNF-α (2, 3.98 vs. 0, 2.66) and IL-10 (13.34, 5.95 vs. 11.92, 2.99) concentrations between SS_u and SS_n. WBV in the SS_u group at 46 sec^-1 and at 230 Sec 1 were 1.9 (95%CI; 1.2, 3.1) and 2.3 (1.2, 4.4) times greater than in the SS_n group. There were no differences in the degree of tissue hypoxia as determined by lightguide spectrophotometry.

Conclusion

Inflammatory, adhesion markers and WBV may be associated with leg ulceration in sickle cell disease by way of inflammation-mediated vasoocclusion/vasoconstriction. Impaired skin oxygenation does not appear to be associated with chronic ulcers in these subjects with sickle cell disease.     

Sucrose Synthase in Developing Maize Leaves: Regulation of Activity by Protein Level during the Import to Export Transition

The maize (Zea mays) leaf is a valuable system to study the sucrose import to sucrose export transition at the cellular level. Rapidly growing and fully heterotrophic cells in the basal part of the young leaf showed a high sucrose synthase (SS) activity. Leaf SS has been purified to homogeneity. By comparison with purified kernel SS isozymes, the leaf SS has been identified as SS₂. SS₁ protein and SS₂ protein were clearly separated by electrophoresis and the two monomers differed in size by 6 kilodaltons. Nevertheless, kinetic parameters of both enzymes were very similar. Immunodetection of SS protein showed that in young heterotrophic tissues SS₂ was a major protein accounting for 3% of the total protein. Concurrent with greening, SS activity decreased and the change of activity was explained by regulation of the protein level. In mature green tissues, which are synthetizing sucrose as evidenced by the presence of sucrose phosphate synthase activity, SS activity was almost completely absent. Results suggested that down regulation of SS₂ enzyme protein level was an early event in the transition from import to export status of the leaf.     

Progress Realized: Trends in HIV-1 Viral Load and CD4 Cell Count in a Tertiary-Care Center from 1999 through 2011

Background

HIV-1 RNA and CD4 cell counts are important parameters for HIV care. The objective of this study was to assess the overall trends in HIV-1 viral load and CD4 cell counts within our clinic.

Methods

Patients with at least one of each test performed by the Infectious Diseases Laboratory from 1999 through 2011 were included in this analysis. By adapting a novel statistical model, log₁₀ HIV-1 RNA means were estimated by month, and log₁₀-transformed HIV-1 RNA means were estimated by calendar year. Geometric means were calculated for CD4 cell counts by month and calendar year. Log₁₀ HIV-1 RNA and CD4 cell count monthly means were also examined with polynomial regression.

Results

There were 1,814 individuals with approximately 25,000 paired tests over the 13-year observation period. Based on each patient''s final value of the year, the percentage of patients with viral loads below the lower limit of quantitation rose from 29% in 1999 to 72% in 2011, while the percentage with CD4 counts <200 cells/µL fell from 31% to 11%. On average annually, the mean HIV-1 RNA decreased by 86 copies/mL and the mean CD4 counts increased by 16 cells/µL. For the monthly means, the correlations (R²) from second-order polynomial regressions were 0.944 for log₁₀ HIV-1 RNA and 0.840 for CD4 cell counts.

Conclusions

Marked improvements in HIV-1 RNA suppression and CD4 cell counts were achieved in a large inner-city population from 1999 through 2011. This success demonstrates that sustained viral control with improved immunologic status can be a realistic goal for most individuals in clinical care.     

Synthesis of thiazol-2-imines from the reduction of single enantiomer 2-imino-thiazolidin-4-ones followed by a spontaneous water elimination

Chiral hemiaminals ( 5-8RR and 5-8SS ) have been synthesized from the corresponding 2-iminothiazolidine-4-ones ( 1-4RR and 1-4SS ) by LiAlH₄ reductions stereoselectively and were then converted to single enantiomer thiazol-2-imines ( 9-12RR and 9-12SS ) by a water elimination reaction. The kinetics of the dehydration reactions which occurred spontaneously both in the solid state and in the solution have been followed by time dependent ¹H nuclear magnetic resonance spectroscopy. The corresponding first order rate constants and free energies of activation values for the conversions have been reported.     

Encephalomyocarditis virus RNA: variations in polyadenylic acid content and biological activity.

Encephalomyocarditis (EMC) viral RNA was isolated from purified virus grown in Ehrlich ascites tumor cells. The viral RNA was found to contain polyadenylic acid [poly(A)] regions that were very heterogeneous in length. Chromatography of the EMC viral RNA on oligo(dT)-cellulose columns separated the RNA into three distinct fractions (peaks 1 to 3). Approximately 20% of the EMC viral RNA appeared as peak 1, 40% as peak 2, and 40% as peak 3. The RNA in each fraction appeared to be intact as shown by co-sedimentation with 35S unfractionated EMC viral RNA in SDS-sucrose density gradients. Approximately 95 to 100% of peaks 1 and 3, and 60 to 70% of peak 2, reappeared at the same elution position after rechromatography on oligo(dT)-cellulose. The RNA in peak 1 contained poly(A) with an average length of 16 nucleotides, peak 2 contained poly(A) with an average of 26 nucleotides, and peak 3 contained an average of 74 nucleotides in its poly(A) region. The distribution in the three fractions, as well as the average length of the poly(A) moieties, was relatively unaffected by changes in the cell suspension medium used during infection. Finally, each of the three viral RNA fractions was assayed for biological activity using an infectious RNA assay on L-cell monolayers. Infectivity of the viral RNA was found to increase with poly(A) length, with peak 3 viral RNA being approximately 10 times more infectious than peak 1 viral RNA.     

Isolation and Purification of Double-Stranded RNA Fragments from Retrovirus RNA

Genomic or high molecular weight RNA of retroviruses consisted of 2 to 5% double-stranded RNA. Fragmentation of genomic viral RNA by limited RNase T₁ treatment before cellulose CF-11 chromatography indicated that 3 to 5% of the viral RNA fragments were eluted as double-stranded RNA. This double-strandedness was in close agreement with the more representative 2 to 3% double-strandedness as determined by RNase T₂ resistance studies performed on genomic and subunit viral RNA. The double-stranded RNA of RNase T₂ treated retrovirus RNA was purified by cellulose CF-11 chromatography.     

Genetic alterations associated with 18F-fluorodeoxyglucose positron emission tomography/computed tomography in head and neck squamous cell carcinoma

BackgroundWe investigated the relationship between genetic alterations and ¹⁸F-FDG PET/CT findings in head and neck squamous cell carcinoma (HNSC).MethodsUsing mRNA-sequences of HNSC samples (480 patients) from the Cancer Genome Atlas (TCGA) portal, gene coexpression networks were constructed via a weighted correlation network analysis (WGCNA) algorithm, and their association with the tumor-to-blood signal ratio on ¹⁸F-FDG PET/CT data (21 patients) was explored. An elastic-net regression model was developed to estimate the PET tumor-to-blood ratio from the gene networks and to derive an FDG signature score (FDG_SS). The FDG_SS was evaluated with regard to clinical variables and general mutational profiles, as well as alterations to oncogenic signaling pathways.FindingsThe FDG_SS values differed across clinical stages (p = 0.027), HPV-status (p< 0.001), and molecular subtypes of HNSC (p< 0.001). Multivariate Cox regression demonstrated that FDG_SS was an independent predictor for overall (p = 0.019) and progression-free survival (p = 0.024). FDG_SS positively correlated with total mutation rate (p = 0.016), aneuploidy (p < 0.001), and somatic copy number alteration scores (p < 0.001). CDKN2A in the cell cycle pathway (q = 0.014) and the TP53 gene in the TP53 pathway (q = 0.005) showed significant differences between high and low FDG_SS patients.ConclusionFDG_SS based on the gene coexpression network was associated with the mutational landscape of HNSC. ¹⁸F-FDG PET/CT is therefore a valuable tool for the in vivo imaging of these cancers, being able to visualize the glucose metabolism of the tumor and allow inferences to be made on the underlying genetic alterations in the tumor.     

RNA from callus cultures and leaves of Nicotiana tabacum

MAK column chromatography has been used to analyse RNA from normal and crown gall callus cultures and leaves of Nicotiana tabacum. To determine the elution behaviour of well-defined DNA-like RNAs with different GC content, complementary RNAs (c-RNA) synthesized on Agrobacterium tumefaciens DNA and crown gall DNA were used. The elution profile of the RNA from all three tissues followed a similar pattern. By salt gradient elution the RNA in the tRNA region showed a remarkably high CMP content which was significantly higher for the normal tissues than for crown gall tissue. RNA from the callus cultures contained more DNA-like RNA (D-RNA) with a higher turnover rate than RNA from leaves. Because of its relatively low poly A content, measured as RNase A + T₁ resistance, as well as its high turnover rate, the salt-eluted D-RNA is thought to be heterogeneous nuclear RNA (Hn-RNA) and not mRNA. RNA molecules that might represent the mRNA population, having intramolecular poly A tracts, were subsequently eluted by a salt gradient, a low salt buffer and with the chaotropic agent guanidine thiocyanate, which removed tenaciously bound (TB-RNA) in two fractions, α and β. Crown gall RNA showed both a different labelling behaviour and a higher poly A content in the α and β fractions compared to the normal tissues. c-RNAs may be eluted at different salt concentrations because of their different GC content. They give rise to a considerable fraction of TB-RNA which in the presence of tobacco leaf RNA was split into fractions similar to α and β. No fraction was found amongst these RNAs which did have intramolecular poly A tracts.     

Nucleotide Composition of RNA hybridized to Homologous DNA from Cells transformed by Avian Tumour Viruses

PART of the evidence which indicates that RNA tumour viruses replicate through a DNA intermediate¹ was the detection of DNA which is complementary to the viral RNA in leukaemic cells transformed by avian myeloblastosis virus (AMV)² and in cells transformed in vitro by avian sarcoma viruses, Schmidt-Ruppin (SR-RSV) and B-77 (ref. 3). If this DNA serves as a template for the viral RNA, it must be a copy of the entire viral genome. One of the necessary requirements for this function is that the homologous DNA has the same nucleotide composition as the viral RNA. In this study, the average base composition of the RNA which had been hybridized to homologous DNA from transformed cells was compared with the base composition of the input viral RNA. Two experimental conditions had to be met: (1) the recovery of all the ribonucleotides which had been hybridized and (2) the absence of partially hybridized ribonucleotide sequences. The first requirement called for the deletion of the treatment of DNA-RNA hybrids with pancreatic ribonuclease fraction A and ribonuclease T₁ which had been used in our previous experiments because such a treatment can cause the non-random loss of hybridized nucleotides⁴. The second requirement called for a hybridization and washing procedure in which only specifically hybridized ribonucleotide sequences would remain bound to the filters. Both of these conditions were met by using fragmented viral RNA and a modified washing procedure which excluded the use of ribonuclease. The results show that the average nucleotide composition of the hybridized RNA is identical to that of the input viral RNA.     

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADH_SS. The ω-hydroxyfatty acid dehydrogenase activity of LADH_SS was of similar magnitude as that of LADH_EE whereas the ω-hydroxysteroid dehydrogenase activity of LADH_SS was considerably higher than that of LADH_EE.     

Adenovirus transcription. V. Quantitation of viral RNA sequences in adenovirus 2-infected and transformed cells.

The concentrations, in copies per cell, of viral RNA sequences complementary to different regions of the genome were determined at 8, 18 and 32 hours after infection of human cells with adenovirus type 2: separated strands of fragments of ³²P-labelled adenovirus 2 DNA, generated by cleavage with restriction endonucleases EcoR1, Hpa1 and BamH1, were added to reaction mixtures at sufficient concentrations to drive hybridizations with infected or transformed cell RNA. Under these conditions, the fraction of ³²P-labelled DNA entering hybrid is directly proportional to the absolute amount of complementary RNA in the reaction.At 8 hours after infection in the presence of cytosine arabinoside, “early” viral messenger RNA sequences are present at a frequency of 300 to 1000 copies per cell. The abundance of early mRNA sequences in different lines of adenovirus 2-transformed rat cells is markedly lower than their concentration in lytically infected cells. Moreover, the abundance of early mRNA in a given transformed rat cell line reflects the number of copies of its template DNA sequences per diploid quantity of cell DNA. After the onset of the late phase of the lytic cycle, the abundance of one early mRNA species, that coding for a single-stranded DNA binding protein required for viral DNA replication, is amplified. Viral RNA sequences complementary to regions of the genome coding for other early mRNA sequences remain at the level observed at 8 hours after infection.Exclusively “late” viral mRNA sequences are present over a range of concentrations, 500 to 10,000 copies per cell, depending on the region of the genome. By 18 hours after infection, the nucleus contains approximately three times as much total, viral RNA as the cytoplasm. The abundant nuclear, viral RNA sequences at 18 hours are transcribed from a contiguous region, 65% of the genome in length. In some cases, viral RNA sequences complementary to mRNA sequences are very abundant in the nucleus. When cytoplasmic and nuclear fractions are mixed and incubated under annealing conditions, some mRNA sequences will anneal with more abundant, anti-messenger nuclear RNA sequences to form double-stranded RNA. Such annealing of nuclear, viral RNA to early, cytoplasmic mRNA sequences probably accounts for the inability to detect, by filter hybridization, certain classes of early mRNA sequences during the late stage of infection.