Small Plaque Size Research Articles

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine.

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.

Involvement of the cytoplasmic domain of the hemagglutinin-neuraminidase protein in assembly of the paramyxovirus simian virus 5.

Efficient assembly of enveloped viruses at the plasma membranes of virus-infected cells requires coordination between cytosolic viral components and viral integral membrane glycoproteins. As viral glycoprotein cytoplasmic domains may play a role in this coordination, we have investigated the importance of the hemagglutinin-neuraminidase (HN) protein cytoplasmic domain in the assembly of the nonsegmented negative-strand RNA paramyxovirus simian virus 5 (SV5). By using reverse genetics, recombinant viruses which contain HN with truncated cytoplasmic tails were generated. These viruses were shown to be replication impaired, as judged by small plaque size, reduced replication rate, and low maximum titers when compared to those features of wild-type (wt) SV5. Release of progeny virus particles from cells infected with HN cytoplasmic-tail-truncated viruses was inefficient compared to that of wt virus, but syncytium formation was enhanced. Furthermore, accumulation of viral proteins at presumptive budding sites on the plasma membranes of infected cells was prevented by HN cytoplasmic tail truncations. We interpret these data to indicate that formation of budding complexes, from which efficient release of SV5 particles can occur, depends on the presence of an HN cytoplasmic tail.

Histopathology of postpercutaneous transluminal coronary angioplasty remodeling in human coronary arteries

Background We studied the histomorphometric correlates of long-term successful coronary balloon angioplasty (percutaneous transluminal coronary angioplasty [PTCA]). Restenosis after PTCA may occur secondary to the failure of compensatory arterial enlargement or post-PTCA arterial constriction. The histopathology of this process in human subjects remains poorly defined. Methods Forty-two coronary segments from 41 patients treated with PTCA 60 ± 58 weeks before death were studied. The histomorphometric findings at the PTCA site were compared with those obtained at a proximal reference site. Results Histologic long-term success was seen in 18 (43%) of 42 arteries. Histologically successful PTCA arteries (PTCA site lumen of ≥50% of the reference lumen) demonstrated a larger acute lumen, smaller plaque size (normalized to the internal elastic lamina area), and thinner adventitia compared with histologic failures. Relative to the reference sites, histologically successful PTCA showed expansion of the external elastic lamina. In contrast, histologic failures showed a reduced external elastic lamina area, suggesting constrictive remodeling. Neointimal area correlated with the extent of internal elastic lamina disruption, but neither variable was related to histologic PTCA success or failure. Conclusions These data provide histomorphometric confirmation of the hypothesis that constrictive remodeling, not neointimal formation, determines the long-term outcome of PTCA. (Am Heart J 1999;138:681-7.)

Site-saturation mutagenesis of the PALTAVETG motif in coxsackievirus A9 capsid protein VP1 reveals evidence of conservation of a periodic hydrophobicity profile.

Enteroviruses possess a highly conserved 9 amino acid stretch of mainly hydrophobic character in the capsid protein VP1. A novel strategy, combining site-saturation mutagenesis and a single-tube cloning and transfection procedure, has been developed for the analysis of this motif in coxsackievirus A9 (CAV-9). Four individual amino acids were separately mutated. Mutagenesis of three of the four positions in CAV-9 resulted in a number of viable but impaired mutant strains, each containing a single amino acid substitution. In contrast, no mutants with amino acid substitutions at leucine 31 were isolated, although three different leucine codons were found among the viruses recovered. Small plaque size was regularly associated with reduced yields of infectious virus and an amino acid substitution at the target site in the viruses isolated from the site-saturated virus pools. From the range of amino acids observed in viable mutants, it was possible to estimate the characteristics that are required at individual amino acid positions. It seems that in the motif studied here, a periodic hydrophobicity profile needs to be conserved. The constraints observed on the ranges of acceptable amino acids presumably reflect the structural-functional requirements that have resulted in the conservation of the motif.

Differences in Plaque Size and VP4 Sequence Found in SA11 Virus Clones Having Simian Authentic VP4

We isolated five stable SA11 clones (TN-S1, TN-S2, TN-L1, TN-L2, and BN-S4) with different plaque sizes from two SA11 stocks. In polyacrylamide gel electrophoresis, the mobilities of the fourth, fifth, and seventh RNA segments of SA11 clones with large plaque size (TN-L1 and TN-L2) were faster than those of clones with small plaque size (TN-S1, TN-S2, and BN-S4). Nucleotide sequence determination of the fourth RNA segment identified a five-amino-acid difference in VP4s between the clones with large and small plaque sizes. The VP4 sequences of two clones with small plaque size (TN-S1 and BN-S4) were the same as the sequence of SA11-SEM reported by K. Nishikawa et al. (1988, J. Virol. 62, 4022-4026), while the VP4 sequences of the clones with large plaque size (TN-L1 and TN-L2) were similar, but not identical, to that reported by D. B. Mitchell and G. W. Both (1989, Nucleic Acids Res., 17, 2122). A single-gene reassortant, K8-L2.4, in which RNA segment 4 was derived from clone TN-L2 of SA11 virus and the other RNA segments were from strain K8, produced large plaques like TN-L2, suggesting that the five-amino-acid difference in VP4 between the clones with large and small plaque sizes might be associated with the difference in plaque size.

The HSV-1 UL45 gene product is not required for growth in vero cells

We have constructed a HSV-1 UL45 null mutant (UL45Δ) by inserting a TK- lacZ cassette into a Bc11 site near the 5′ end of the UL45 gene. A polyclonal antiserum produced to an Escherichia coli trpE:UL45 fusion protein was used to show that an 18-kDa polypeptide corresponding to the predicted UL45 gene product was produced in HSV-1 strain KOS-infected Vero cells but was not detected in UL45Δ-infected Vero cells. The absence of the 18-kDa protein had only a slight effect on viral growth in cell culture, indicating that the UL45 gene product is not essential for growth in Vero cells. However, the burst size of UL45A was smaller than HSV-1 KOS in Vero and HeLa cells. UL45Δ also had a smaller plaque size and an altered plaque morphology

A single point mutation of Ala-25 to Asp in the 14,000-Mr envelope protein of vaccinia virus induces a size change that leads to the small plaque size phenotype of the virus.

The molecular defect responsible for a structural and functional abnormality of the 14,000-molecular-weight (14K) envelope protein of vaccinia virus has been identified. Through DNA sequence analysis of the entire 14K gene from wild-type vaccinia virus and three vaccinia virus mutants, a single base change of C to A was found that resulted in the substitution of Asp for Ala-25. This mutation is responsible for protein size abnormality, as documented by cell-free translation in a rabbit reticulocyte lysate of in vitro mRNA transcripts. In addition, through marker rescue experiments we show that this mutation is responsible for the small plaque size phenotype of vaccinia virus mutants. The structural consequence of the point mutation is a possible turn in an alpha-helix domain with destabilization of a hydrophobic interaction at the N terminus, resulting in monomers and trimers of vaccinia virus 14K protein with decreased electrophoretic mobilities. The functional consequence of the point mutation is a reduction in virulence of the virus.

Modification of foot-and-mouth disease virus after serial passages in the presence of antiviral polyclonal sera

Foot-and-mouth disease virus (FMDV) shows a remarkable antigenic variability. Like other RNA viruses, this virus has a high rate of mutation. It has been proposed that selection exerted by the host's antibodies could play a major role in the rapid evolution of FMDV. The present work reports the selection of FMDV antibody-resistant populations (Nr), after serial passages of cloned FMDV A 24 Cruzeiro strain on secondary monolayers of bovine fetal kidney cells in the presence of subneutralizing antiviral polyclonal sera (APS). After a limited number of passages under selective pressure, the virus population showed the following characteristics: (1) increased resistance to neutralization by APS; (2) altered electrophoretic mobility of structural viral proteins (VP1); (3) remarkable plaque size reduction, (4) a pronounced thermosensitivity (ts); and (5) decreased pathogenicity for mice, in both uncloned and cloned small plaque size populations. This indicates that FMDV populations under antibody pressure in vitro, have acquired, in addition to expected characteristics of natural FMDV variants (resistance to neutralization and altered viral structural proteins), phenotypic markers which correspond to attenuated, less virulent variants.

Japanese encephalitis virus live-attenuated vaccine, Chinese strain SA14-14-2; adaptation to primary canine kidney cell cultures and preparation of a vaccine for human use

The Japanese encephalitis (JE) live-attenuated vaccine virus clone SA14-14-2 was adapted to grow in primary canine kidney (PCK) cell culture, and vaccine seeds and a first lot of vaccine were prepared in these cells. Characterization of the PCK-grown virus by various laboratory and animal tests indicated that passage in PCK did not result in detectable phenotypic or genome changes for this virus clone. Markers of attenuation included small plaque size, lack of intracerebral virulence for weanling mice, minimal neurovirulence for rhesus monkeys and a distinct nucleotide pattern compared to the parent SA14 non-attenuated virus. In addition, the seeds and vaccine were free of any detectable adventitious microbial agents that would render these materials unsafe for human immunization. Small-scale clinical trials of the JE SA14-14-2 PCK vaccine can now proceed to test the human safety of this product.

Isolation, Complementation and Partial Characterization of Temperature-sensitive Mutants of the Baculovirus Hyphantria Cunea Nuclear Polyhedrosis Virus

Summary Twelve temperature-sensitive mutants were isolated from Hyphantria cunea nuclear polyhedrosis virus grown in Spodoptera frugiperda cells and were sorted into four groups by their properties in plaque assays at the non-permissive temperature (32 °C). The phenotypes of the four groups were as follows: (i) failure to make polyhedra, (ii) few polyhedra formed, (iii) reduced plaquing efficiency, (iv) small plaque size. Ten mutants had reduced plaque size and polyhedra formation at 32 °C. One mutant formed plaques without polyhedra, had a reduced infectious virus titre at 32 °C and also showed a defect in late gene function. Two mutants formed small plaques with few polyhedra and were temperature-sensitive with respect to production of extracellular non-occluded virions at 32 °C. Other phenotypes were also distinguished. The formation of polyhedra by all the mutants was 2 to 4 h faster at 32 °C than at 25 °C. After temperature shift-up from 25 °C to 32 °C at 12 h post-infection polyhedron formation was still 2 to 4 h faster. Complementation analyses based on polyhedron formation in double infections at 32 °C distinguished four complementation groups.

Isolation and characterization of attenuated mutants of vaccinia virus

Attenuated variants of vaccinia virus with two specific and stable genetic markers were obtained after long-term passage of persistently infected Friend erythroleukemia (FEL) cells. The genetic markers were an 8-MDa deletion on the left HindIII-C terminus of the viral genome and sequence alterations localized in the middle of the HindIII-A DNA fragment. This latter genetic marker led to small plaque size phenotype of these variants. The mode of replication of these variants in tissue culture cells and their virulence in mice were analyzed. In cultured cells, these variants have greatly reduced virus yields in cell lines of different origins. These variants penetrate into cells, synthesize early and late viral proteins, and replicate their DNA with about the same efficiency as wild-type virus. The defect of these variants appears at some step during virus maturation. When groups of BALB/c mice were inoculated intraperitoneally (ip) with these variants, 50% of the mice survived with ⩾ 1 × 10 9 plaque-forming units (PFU) as opposed to about 50% survival for mice inoculated with 1 × 10 6 PFU of wild-type virus. Mice inoculated with these variants were fully protected when challenged ip with lethal doses of wild-type virus. The reduced virulence of these variants correlated with the 8-MDa deletion; in addition, the plaque size phenotype marker contributes to a further decrease of the virulence of vaccinia virus. Due to their limited virus production and protective immune response, these variants may be potentially useful as vaccines.

A 14K envelope protein of vaccinia virus with an important role in virus-host cell interactions is altered during virus persistence and determines the plaque size phenotype of the virus

The phenomenon of genetic variability and attenuation or virulence of poxviruses is poorly understood. We have identified mutants of vaccinia virus from untreated and interferon (IFN)-treated persistently infected Friend erythro-leukemia (FEL) cells that have major alterations in the size of a virus structural protein. This protein is part of the virus envelope as documented with specific monoclonal antibody (mAbC3). This protein, under reducing conditions, has a molecular weight of about 14,000 (14K) Da in wild-type virus but gained 1.5 K in mutants from untreated, persistently infected cells and about 0.5K in mutants from IFN-treated persistently infected cells. Under nonreducing conditions, this protein forms covalently linked oligomers which also differ in size between wild-type and mutant viruses. The 14K protein elicits humoral immune response as assessed by immunoblots of two-dimensional SDS-PAGE analysis using rabbit anti-vaccinia serum. Two molecular forms of the 14K protein with different isolectric points were found only in mutants from untreated, persistently infected cells. Protein modifications were the result of DNA sequence alterations in the virus population since the 15.5K protein could be reverted to 14K after marker-rescue with the cloned 14K encoding gene. We provide direct evidence that changes in size of the 14K envelope protein are responsible for the small plaque size phenotype of these variants. From our previous studies ( J. F. Rodriguez, R. Janeczko, and M. Esteban, 1985. J. Virol. 56, 352–356; J. F. Rodriguez, E. Paez, and M. Esteban, 1987, J. Virol. 61, 395–404) and those described here, we established that the 14K protein plays an important role in virus-host cell interactions. This is because this protein is part of the virus envelope, is highly antigenic, elicits neutralizing antibodies, has a role in virus penetration acting in cell fusion, is involved in the virus plaque size phenotype and is highly conserved among Orthopoxviruses. In addition, because mutants with altered 14K protein that have gained 1.5K in size are attenuated ( S. Dallo and M. Esteban, 1987, Virology 159, 408–422), it suggests that a modified form of this protein contributes to the decrease of the virulence of vaccinia virus.

Development of an attenuated strain of chikungunya virus for use in vaccine production.

An attenuated chikungunya (CHIK) virus clone was developed for production of a live vaccine for human use. CHIK strain 15561 was subjected to 18 plaque-to-plaque passages in MRC-5 cultures before CHIK 181/clone 25 was selected as vaccine seed based on homogeneous small plaque size, suckling mouse avirulence, reduced monkey viraemia and genetic stability. Oligonucleotide mapping demonstrated differences between parent and clone. Vaccine (pilot-lot production) elicited neutralizing antibody and protected mice and rhesus monkeys against challenge. After challenge, viraemias were absent in vaccinated monkeys. Vaccine was then produced and tested in accordance with governmental regulatory requirements of human use.

Formation, characterization and interfering capacity of a small plaque mutant and of incomplete virus particles of infectious bursal disease virus

Serial undiluted passages of infectious bursal disease virus in chick embryo cells were accompanied by a von Magnus type fluctuation of infectivity in viral harvests and a gradual decrease of plaque size. From the 9th undiluted passage on, the whole virus population consisted of small plaque-forming virus. The small plaque size remained constant when subsequent infections were carried out at low multiplicities. Small plaque virus interfered with the replication of large plaque standard virus. The small plaque/low yield mutation favoured the generation of defective particles which could be separated from complete particles by their lower densities in CsCl-gradients, where six fractions became visible and could be analyzed separately. Most of the defective virus particles had lost the larger of their two dsRNA segments and showed an aberrant protein composition. They had a very low residual infectivity and were also able to interfere with the replication of complete virus.

Fetal rhesus monkey lung cells can be grown in serum-free medium for the replication of dengue-2 vaccine virus.

Serum-free media were developed to grow diploid fetal rhesus monkey lung (DBS-FRhL-2) cells and to propagate dengue-type 2 virus vaccine strain PR-159 (dengue-2 vaccine virus). Vitamins, amino acids, growth factors, hormones and other organic compounds, and inorganic salts were substituted for fetal bovine serum. The composition of the medium that was optimal for growth of DBS-FRhL-2 cells differed from medium optimal for the propagation of dengue-2 vaccine virus. Insulin, epidermal growth factor, fibroblast growth factor, and platelet-derived growth factor were required for DBS-FRhL-2 cell proliferation in serum-free medium but were inhibitory for virus propagation. Adenosine, cytidine, guanosine, uridine, and thymidine, each at 0.01 mM concentration, were necessary as medium supplements to obtain a high yield of dengue-2 vaccine virus in DBS-FRhL-2 cells under serum-free conditions. DBS-FRhL-2 cells grown in serum-free medium produced dengue-2 vaccine virus with yields similar to those of cells grown in the presence of serum. Dengue-2 vaccine virus obtained under serum-free conditions retained its phenotypic markers such as temperature sensitivity and small plaque size.

Construction and characterization of viable deletion mutants of simian virus 40 lacking sequences near the 3' end of the early region.

Five viable deletion mutants of simian virus 40 (SV40) were prepared and characterized. These mutants lack 15 to 60 base pairs between map positions 0.198 and 0.218, near the 3' end of the early region of SV40 and extend further into the body of the A gene, encoding the large T antigen, than previously described deletion mutants. These mutants were isolated after transfection of monkey kidney CV-1p cells with full-sized linear DNA prepared by partial digestion of form I SV40 DNA with restriction endonucleases HinfI or MboII, followed by removal of approximately 25 base pairs of DNA from the 5' termini using lambda-5'-exonuclease and purification of the DNA in agarose gels. Based on camparisons of the DNA sequence of SV40 and polyoma virus, these mutations map in the 19% of the SV40 A gene that shares no homology with the A gene of polyoma virus. The mutations exist in two different genetic backgrounds: the original set of mutants (dl2401 through dl2405) was prepared, using as a parent SV40 mutant dl862, which has a deletion at the single HpaII site (0.725 map unit). A second set (dl2491 through dl2495) contains the same deletions in a wild-type SV40 (strain SV-S) background. Relative to wild-type SV40, the original mutants showed reduced rates of growth, lower yields of progeny virus and viral DNA, and smaller plaque size; in these properties the mutants resembled parental dl862, although mutant progeny yields were usually lower than yields of dl862, suggesting a possible interaction between the two deletions. The second set of mutants had growth properties and progeny yields similar to those of wild-type SV40; however, Southern blotting experiments indicated that viral DNA replication proceeds at a slightly reduced rate. All of the mutants transformed mouse NIH/3T3 cells and mouse embryo fibroblasts at the same frequency as wild-type SV40. Mutants dl2402, dl2492, and dl2405 consistently produced denser and larger foci in both types of cells. All mutants directed the synthesis of shortened large T antigens. Adenovirus helper function was retained by all mutants.

A simplified plaque assay system for measles virus

A plaque assay for measles virus in Vero cells under a carboxymethylcellulose overlay is described. Due to the small plaque size, 24 well plates can be used giving an assay applicable to large numbers of samples while retaining the precision of traditional plaque assay systems. In addition to precise quantitative values for infectivity, the method allows for monitoring of qualitative plaque type and provides a permanent record of the test. Plaque and TCD 50 assay systems were found to give comparable results in tests where conditions of virus adsorption were similar. The method has been successfully applied to the stability testing of measles vaccines and is applicable to other aspects of vaccine standardization.

Production of temperature-sensitive and pathogenic virus fromAedes albopictus cells (Singh) persistently infected with chikungunya virus

When A. albopictus, clone C6/36, cells were infected with chikungunya (CHIK) virus, high virus yield accompanied by a cytopathic effect in the acute stage of infection was followed by a relatively low yield of virus over a long period of time. Virus produced from persistently infected cultures became gradually of smaller plaque size and more temperature-sensitive; however, such virus still retained pathogenicity for suckling mice even after one year of infection. When the persistently infected cells were subcultured, a dissociation was observed between the time course of cell growth and that of virus production, suggesting some intracellular mechanisms that turn off virus production. The greater part of the interference against CHIK virus by the culture medium of the persistently infected cells appeared to be mediated by the infective virus in the medium. The infective virus was easily removed from the persistently infected cells either by subculture or by cloning in the presence of anti-CHIK serum, yielding cured cultures or virus-negative clones.

Effect of 5-Iodo-5′-Amino-2′,5′-Dideoxyuridine on Varicella-Zoster Virus In Vitro

The antiviral effect of the nucleoside analog 5-iodo-5'-amino-2',5'-dideoxyuridine (AIU) was tested with three isolates of varicella-zoster virus (VZV). AIU concentrations of 10 to 800 muM (3.5 to 288 mug/ml) reduced the number of plaques produced by VZV-infected cells and cell-free VZV from approximately 30 to 95%. Smaller plaque size was also observed in the presence of AIU. AIU was less effective than arabinofuranosylthymine in reducing VZV-induced plaques since as little as 5 mug of arabinofuranosylthymine per ml completely blocked plaque formation by cell-free VZV. Toxicity assays with human diploid embryo fibroblast cells were also carried out. Drug concentrations as high as 800 muM were not toxic to human diploid embryo fibroblast cells as determined by radiolabeling of cell deoxyribonucleic acid, ribonucleic acid, and protein.

Studies of arboviruses in Southwestern Venezuela: I. Isolations of Venezuelan and Eastern Equine Encephalitis viruses from sentinel hamsters in the Catatumbo region.

The purpose of this report is to describe isolations of Venezuelan (VEE) and Eastern (EEE) Equine Encephalitis virus made in the lowland moist tropical forest of the Catatumbo region on the southwestern part of the State of Zulia, Venezuela. We have isolated four strains of EEEV from sentinel hamsters exposed at Caño Mocho and Madre Vieja sites in 1973 and 1974, and three strains of EEEV in Hacienda (Hda.) Las Nubes in 1975. Both viruses were recovered during silent interepidemic periods and we believe these viruses are maintained in this region in sylvatic conditions. The recovered virus strains were detected within 24 to 48 hours, both in SMB and Vero Cell monolayer cultures and the sentinel hamsters yielded virus infectivities up to 10(4) PFU ml. Our VEEV isolate (IVIC PAn 23645-47), recovered during the silent interepizodemic period had an elution profile on the hydroxylapatite column corresponding to that of a I-D (VEEV-3880) or a I-E (VEEV-63A216) 'enzootic' subtype. However, considering other in vitro criteria (KHI; HA pH 5.8-6.0; small plaque size in Vero monolayers with suitable overlay media), this later and other previous isolates had some very distinct properties of the 'epizootic' strains. Thus, the evidence suggests that in Venezuela the VEEV cycle in nature is maintained either by the so called 'enzootic' and/or 'epizootic' virus types, or the virus population of the isolates have particular in vitro properties which do not correlate to the virulence markers in vivo. We consider this important question must be further clarified, and in addition, the isolation of three strains of EEEV are reported; this is the first report of the presence of this virus in Venezuela. Although the EEEV isolates may be of the South American type, they must be considered as potentially dangerous in the case of outbreaks.