Characterization of rabies viruses recovered from persistently infected BHK cells.

Persistent rabies virus infection with a cyclic rising and falling pattern was easily initiated in BHK cells. In striking contrast with the same chronic infection established by T. J. Wiktor and H. F. Clark ((1972). Infect. Immun.6, 988–995), this virus and cell interaction was not mediated by interferon, and the infected cells were not resistant to a heterologous virus challenge. The persistently infected cells produced small-plaque viruses (SPV) and, after a number of subcultures, these SPV replaced entirely the wild large-plaque viruses (LPV). Due to the marked cytopathic effects (CPE) on BHK cells, SPV was less efficient than LPV for the establishment of viral persistence.A fraction containing defective interfering (DI) rabies virus particles was obtained by density gradient centrifugation of virus stocks from the persistently infected cells. The DI particles could be quantitatively assayed to the degree that they suppressed the CPE by the standard rabies virus. Cyclic production of DI particles in the persistent infection was demonstrated by this method. This evidence plus the fact that the homologous virus resisted superinfection indicate that the DI particles of rabies virus play a major role in the establishment and maintenance of a persistent state of infection in vitro.     

Proviral load and expression of avian leukosis viruses of subgroup C in long-term persistently infected heterologous hosts (ducks)

Summary.  Proviral DNA load and expression of avian leukosis viruses of subgroup C (ALV-C) in ducks infected in mid embryogenesis were studied using quantitative PCR, RT-PCR, in situ hybridization employing ALV-specific riboprobe, and immunohistochemistry. A group of long-term surviving, non-reviremic ducks was selected for the study and compared to control reviremic animals in order to obtain information about persisting retroviruses in different duck tissues. A widespread distribution of proviruses in the tested tissues was found, but the proviral load was significantly lower in non-reviremic in comparison to reviremic animals. The only exception were brain and blood cells, in which no significant difference in the quantity of integrated proviruses was found between both categories of ducks, thus indicating an exceptional position of the brain and blood cells among all tested tissues. Contrary to reviremic, the proviruses were not transcribed in non-reviremic ducks, with the exception of brain and thymus. In the majority of non-reviremic ducks viral RNA was revealed in the brain, but no infectious virus could be recovered from this tissue. The opposite situation was observed in the thymus, where infectious virus was recovered but viral RNA remained below the detection limit of the assay. As revealed by in situ analysis, infected cells were either disseminated or focally distributed in tissues. From the long-term follow up of ALV-C in intraembryonally infected ducks we conclude that this model is suitable for the study of retrovirus persistence accompained both by the presence and absence of reviremias. The possible consequences of transmission and long-term persistence of retroviruses in the heterologous host for retroviral evolution are discussed. Received November 10, 1998 Accepted February 11, 1999     

Dependence of the antiviral activity of the poly(G).poly(C) complex on the size of the continuous poly(C)segments

Modification of poly(C) by various frequency treatment with adenosine non-complementary to guanosine has produced poly(G) X poly (C.A) complexes with continuous double-stranded areas the length of which is determined by C/A ratio. Studies of the antiviral activity of poly(G).poly(C,A) complexes with C/A from 10:1 to 90:1 and poly(G).poly(C) in vesicular stomatitis virus-infected chick embryo cell cultures and in experimental tick-borne encephalitis of mice demonstrated that the maximum activity is achieved at an average lengths of double-stranded areas of 90 nucleotide pairs. At the same time, a low but statistically significant antiviral activity is observed at a length of double-stranded areas of 10-30 nucleotide pairs.     

The size and location of the poly(A) tract in EMC virus RNA.

Encephalomyocarditis (EMC) virus RNA, selected by its affinity for oligo(dT)-cellulose, contains poly(A) of size : (i) about 14 nucleotide residues long, based on the percentage of radioactivity in the RNA resistant to digestion by a mixture of pancreatic and T1 RNases; (ii) about 15 residues long, as measured by the ratio of the amount of terminal adenosine to internal adenylic acid in isolated poly(A); and (III) in the range 12 to 45 residues, the majority of tracts being about 16 to 18 residues long, based upon electrophoretic mobility on polyacrylamide gels using poly(A) molecules of known size as mol. wt. markers. The poly(A) appears to be located at the 3'-terminus of the virus genome since the tract, liberated by digestion with a mixture of pancreatic and T1 RNases, was shown by compositional analysis to contain a non-phosphorylated 3'-terminus and only adenine residues. The size heterogeneity in the poly(A) tracts revealed by gel electrophoresis is also consistent with a terminal location. Comparison of our data for EMC virus with published data for other picornaviruses suggests that the sizes of poly(A) tracts in polio- and Mengovirus RNA have been overestimated; poly(A) tracts in cardioviruses appear to be smaller than those in poliovirus; the minimum size of poly(A) required for full infectivity of picornavirus RNA has also been overestimated; a tract of at least 13 adenine residues long is required for full infectivity of EMC virus RNA.     

The genomic RNA of mengovirus: I. Location of the poly(C) tract

Strains of λ phage containing a mutated cII, cIII, or cy gene in addition to a cro^? mutation were constructed. Each single mutant and the double mutants were analyzed for growth behavior and for expression of specific early and late λ proteins. λc1857cro 16 is unable to grow well at 30° and at 43°. This strain is incapable of turning off early genes (of the b region, of the P_L and P_R operons, and the cU and int genes) and is defective in the expression of late functions. The clear mutations permit phage growth at 30°. They relieve the inhibitory effect conferred by the cro^? mutation on phage growth at 43° to different extents. Our results also indicate that the cII and cIII proteins do not necessarily act at y in the regulation of late functions.     

Evaluation of the size of the continuous poly(G) site necessary for the biological activity of the poly(G).poly(C) complex

On the basis of synthesis of a series of poly(G, A).poly(C) copolymers with changing G:A ratio from 15:1 to 90:1 and trials of their biological activity in comparison with poly(G).poly(C), the size of poly(G) in it was evaluated within the range of a continuous double-stranded area necessary for the activity. The antiviral activity close to that of poly(G).poly(C) in experimental tick-borne encephalitis of mice and vesicular stomatitis virus infection of chick embryo cells was found only in poly(G,A).poly(C) complexes with a G:A ratio equal to or higher than 90:1. Consequently, the high activity of poly(G).poly(C) is present at an average length of poly(G) equal to 90-100 nucleotides within the limits of the continuous double-stranded area.     

Genotyping of bovine viral diarrhea viruses (BVDV) isolated from cattle in Sicily

Bovine Viral Diarrhea–Mucosal Disease (BVD–MD) is a widely spread infectious disease that causes important economic losses in farms. Several epidemiological studies indicate a high genetic heterogeneity among Bovine Viral Diarrhea Virus (BVDV) strains circulating in Italy. The aim of this study was to investigate the genotypes of BVDV in Sicily, a region in the South of Italy. For this purpose, 17 BVDV strains collected from cattle breed in Sicily between 2005 and 2008 were genetically typed by sequencing of the 5′-untraslated region (5′-UTR) of the viral genome. In this study, phylogenetic analysis showed that all 17 examined strains were clustered within the BVDV genotype 1. Particularly, 14 of them were clustered with the BVDV-1b subgroup, while the remaining three strains were clustered with the BVDV-1e. Moreover, the restriction analysis indicated a bovine origin for all of the 17 strains typed in this study. These results could be useful to carry out an epidemiological survey and to create vaccines that protect cattle against BVDV different subgroups.     

Consistent change in the B-C loop of VP2 observed in foot-and-mouth disease virus from persistently infected cattle: implications for association with persistence

The mechanisms of foot-and-mouth disease virus (FMDV) persistence are poorly understood. It is thought the existence of viral quasispecies that encompass sub-populations with varying survival competencies and antigenicities may play some role in the maintenance of virus in persistently infected animals. By analyzing nucleotide sequences encoding the viral VP2 protein in oesophageal-pharyngeal fluid (probang) samples from cattle at different stages of infection, the significance of any amino acid changes in relation to persistence was investigated. Twenty-two experimentally infected cattle (including six carriers) from three animal experiments with FMDV type O UKG34/2001 were studied. Comparison of VP2 sequences in these samples with the inoculum sequence revealed a consistent change in the B-C loop in FMDV from persistently infected cattle. Residue 2079 changed from Y to H in five carrier animals and residue 2080 changed from A to Q in one carrier from 14 days post-infection onward. In contrast, there were no changes evident in any of the non-carriers up to 28 days post-infection. The results indicate that a substitution change in the B-C loop of VP2 may be associated with persistent FMDV infection in cattle.     

聚乳酸-羟基乙酸输尿管支架植入后犬输尿管周围的组织学变化

背景：泌尿系统组织工程支架不仅需要生物相容性良好的生物材料,而且一定要利于组织周围细胞的生长。 目的：制备聚乳酸-羟基乙酸共聚物可降解输尿管支架,观察其植入后犬输尿管周围组织学变化。 方法：制备纳米聚乳酸-羟基乙酸共聚物输尿管支架,并以多聚赖氨酸对支架进行交联、改性,将交联后支架截成长约0.8 cm小段,植入犬损伤输尿管中进行体内观察实验。 结果与结论：①支架制备：支架具有纳米结构,孔隙率约90%,孔径(30±18) µm,多聚赖氨酸交联改性后纤维表面略显粗糙。②支架变化：支架植入30 d时已完全失去原始形态,与周边组织融合,可见裂解小块。③支架植入后输尿管周围组织学变化：植入后15 d炎症表现最为明显,主要是移行上皮脱落,肌层结构被破坏,固有层水肿明显;30 d后,炎症已经明显好转,但组织结构依然不规则;植入后45 d,输尿管全层组织基本恢复正常,组织结构成规则分布。说明聚乳酸-羟基乙酸共聚物输尿管支架具有良好的组织相容性,符合泌尿系统组织工程支架的要求。      

Transmission in vitro of hepatitis C virus from persistently infected human B-cells to hepatoma cells by cell-to-cell contact

Virus cell-to-cell spread has been reported for many different viruses and may contribute to pathogenesis of viral disease. The role played by cell-to-cell contact in hepatitis C virus (HCV) transmission was studied in vitro by cell co-cultivation experiments. A human lymphoblastoid B-cell line, infected persistently with HCV in vitro (TO.FE(HCV)), was used as HCV donor [Serafino et al., 2003]; recipient cells were the human hepatoma HepG2 cell line. Both cell types were co-cultured for 48 hr to allow the cell-to-cell contacts. The hepatoma HepG2 cells are not permissive to free-virus infection, but they were infected successfully using TO.FE(HCV) cells as source of virus. The kinetics of viral RNA synthesis and the percentage of infected cells were compared in cell-mediated-and cell-free-viral infection. After co-cultivation, a consistent proportion of hepatoma cells replicated HCV and stably expressed viral antigens. Virus produced was infectious as demonstrated by the ability to reinfect fresh B-cells. This cell model shows that permissiveness to HCV infection can be achieved in vitro in non-permissive hepatoma cells by direct cell-to-cell contacts with infected human B-cells. This mechanism of virus spread may also play a pathogenic role in vivo.     

Macroporous poly(L-lactide) of controlled pore size derived from the annealing of co-continuous polystyrene/poly(L-lactide) blends

A detailed study on the static annealing of co-continuous polystyrene/poly(L-lactide) (PLLA) blends is presented. The effects of temperature, time at temperature, viscosity of the phases and interfacial modification on the coarsening of the blend are discussed. In this paper, polystyrene and PLLA are blended at compositions of 50/50 and 60/40 to form co-continuous morphologies. These co-continuous morphologies are coarsened under quiescent annealing conditions, and the subsequent removal of the polystyrene phase leaves a macroporous PLLA structure. The microstructure is analyzed using three different techniques: the BET nitrogen adsorption technique, mercury intrusion porosimetry and SEM combined with image analysis. It is shown that static annealing can be used to generate a series of co-continuous networks with controlled pore sizes ranging from 1 to hundreds of microns. A non-linear pore size growth rate is observed for these systems due to the degradation of PLLA and this study indicates that controlled degradation can be used as an additional tool for morphology control. Compatibilized polystyrene/PLLA blends demonstrate significantly reduced coarsening effects due to the reduction of interfacial tension. The coarsening rate of the co-continuous structure was examined in terms of the pore size, R and this growth rate is discussed in terms of a previously proposed coarsening mechanism. This approach is a route towards the preparation of a macroporous PLLA structure with pore sizes in the range required for scaffolds for tissue regeneration.     

Biochemical characterization of an aphthovirus type 0(1) strain campos attenuated for cattle by serial passages in chicken embryos

The biochemical properties of a virulent and an attenuated strain of foot-and-mouth disease virus (FMDV) Type 0(1) Campos (0(1)C) were compared in order to establish differences that could account for their altered biological functions. The avirulent strain (0(1)C-O/E) was derived from the virulent strain 0(1)C by serial passages in chicken embryos. Analysis of the RNase T1-generated oligonucleotides of the viral RNA through one- and two-dimensional (2D) gel electrophoresis (fingerprints) revealed a few changes in the genome structure of the 0(1)C-O/E strain compared to the wild type strain. In addition there was a significant decrease in the length of the poly(C) rich tract of the 0(1)C-O/E RNA. All virion structural proteins, except VP4, their precursors, and the viral RNA polymerase (p56a) show charge differences. In addition a significant decrease in the apparent molecular weight of polypeptide p100 (primary translational product from the 3' end region of the genome) of the attenuated strain was observed.     

Relationship between large granular lymphocytes and NK-1.2+ cells from normal and poly(inosinic:cytidylic acid) (poly(I:C]-treated mice

The present work analyzes the relationship between large granular lymphocytes (LGL), NK-1.2+ cells, and natural killer (NK) activity of C3H/HeN mice. Different hematic cell fractions were obtained according to their nylon-wool adherence and density on Percoll gradients. NK-1.2+ cells (8% of nucleated cells) were more numerous than LGL (3% of nucleated cells) in the input blood population. Eighty-five percent of LGL were recovered from the sorted NK-1.2+ cell fraction. After incubation on nylon-wool column, 63% of LGL and 36% of NK-1.2+ were eluted in the nonadherent fraction. Eighteen percent of NK-1.2+ cells were recovered from the most adherent elutable cell fraction. After the discontinuous Percoll gradient most LGL were present in the low-density fractions while 20% of NK-1.2+ cells were recovered from the highest-density fraction. NK activity was significant both in the nylon-wool-nonadherent and -adherent fractions. After the Percoll gradient most NK activity was present in the low-density fractions. In the present experimental conditions treatment poly(inosinic:cytidylic acid) (poly(I:C] did not increase the numbers of LGL and NK-1.2+ cells either in the blood or in the spleen. However it increased significantly the NK activity of the input cell populations and of the nonadherent and low-density fractions. Similarly, exposure of specific pathogen-free (SPF) mice to non-SPF conditions stimulated NK cytotoxicity but did not alter the percentage of LGL in the blood or in the spleen. Poly(I:C) treatment induced a shift of LGL and NK-1.2+ cells toward the low-density fractions. In poly(I:C)-treated mice images of granule secretion from LGL were detected. Taken together, the present results indicate that LGL and NK-1.2+ cell populations do not totally overlap. Moreover subpopulations of LGL and NK-1.2+ cells can differ in NK activity, morphology, density, adherence to nylon wool, and response to poly(I:C).     

Controlled delivery of taxol from poly(ethylene glycol)-coated poly(lactic acid) microspheres.

The development of injectable microspheres for sustained drug delivery to the arterial wall is a major challenge. We demonstrated the possibility of entrapping an antiproliferative agent, taxol, in poly(ethylene glycol) (PEG)-coated biodegradable poly(lactic acid) (PLA) microspheres with a mean diameter of 2-6 microm. A solution of taxol and PLA dissolved in an acetone/dichloromethane mixture was poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA] with stirring with a high-speed homogenizer for the formation of microspheres. Taxol recovery in PLA-PEG microspheres was higher (61.2 +/- 2.3%) than with PVA-based (41.6 +/- 1.8%) preparations. An analysis by diffuse reflectance infrared Fourier transform spectroscopy revealed that PEG was incorporated well on the PLA microsphere surface. Scanning electron microscopy revealed that the PEG-coated PLA microspheres were spherical in shape and had a smooth surface texture like those of PVA-based preparations. The amount of drug release was much higher initially (25-30%); this was followed by a constant slow-release profile for a 30-day period of study. This PEG-coated PLA microsphere formulation may have potential for the targeted delivery of antiproliferative agents to treat restenosis.     

Alterations in the tRNA''s of Escherichia coli recovered from lethally infected animals.

Escherichia coli grown in chemically defined iron-deficient media or in fluids containing the iron-binding proteins transferrin, lactoferrin, or ovotransferrin have well-characterized alterations in the chromatographic properties of tRNA's containing the modified nucleoside 2-methylthio-N6-(delta2-isopentenyl)-adenosine. The present work shows that similar tRNA alterations occur in E. coli O111 recovered from the peritoneal cavities of lethally infected guinea pigs and rabbits. Adding iron to these in vivo-grown bacteria resulted in the rapid conversion of chromatographically abnormal tRNA's to the normal species. The work strongly suggests that host iron-binding proteins, present in mucosal and other secretions, can affect the metabolism of invading organisms. The idea that the tRNA alterations are connected with the adaptation of E. coli to growth under the iron restricted conditions imposed by iron-binding proteins in tissue fluids, and thus with bacterial pathogenicity, is therefore made particularly attractive.     

Bovine Herpesvirus type-4 (BHV-4) persistently infects cells of the marginal zone of spleen in cattle

Bovine Herpes virus type 4 (BHV-4) has the ability to persist during long post-infection periods in spleen and other lymphoreticular tissues of cattle and laboratory rabbits. Our previous studies indicated that splenic macrophages are the main reservoir of this persistent herpesvirus infection in rabbits. Now we report the use ofin situhybridization (ISH) and cell separation methods to characterize the cellular localization of persistent BHV-4 in cattle. Using cloned sub-genomic probes of BHV-4 DNA labelled with³⁵S, we detected BHV-4 nucleic acids in cells of the marginal zone of spleen from persistently infected cattle and rabbits. In addition, cell separation studies indicated that a non-T, non-B cell population of the bovine spleen harbours BHV-4. This association requires cell integrity andin vitroco-cultivation for re-expression of the persistent virus. We were also able to detect BHV-4 by explantation/co-cultivation from several other tissues of cattle including trigeminal ganglia, urinary bladder, kidney, lung and several lymphoid tissues including lymph nodes and thymus.     

Effect of the size of the continuous poly(G) site in poly (G, A).poly (C) complexes on their interferon-inducing activity and their capacity to stimulate the development of immunity

It was established that the level of interferon-inducing activity of poly(G90A1).poly(C) complex in cell cultures and in mice was comparable to that of poly(G).poly(C). As the size of the continuous sites of poly(G) in the purine strand in poly(G, A).poly(C) complexes decreased to 60 and 28 nucleotides, the interferon-inducing activity decreased progressively, was still marked, or approached the zero at G:A ratios equal to 17:1 or 10:1, respectively. All this indicates that the cell receptors responsible for switching on of the induction mechanisms for interferon synthesis still recognize the stimulus 17 and possibly less so stimulus 10 of successively located guanosine nucleotides complementary to poly(C) and provide for the highest interferon production level when their number is equal to or exceeds 90-100. With the exception of poly(G10A1).poly(C) in which the interferon-inducing activity did not exceed its detection threshold, all complexes enhanced noticeably or markedly specific immune response in mice to tick-borne encephalitis after immunization with inactivated unadsorbed tissue culture vaccine against this infection. The level of this immunostimulating activity correlated irregularly with the intensity of their interferon-inducing activity.