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PubMed1990

Electrophoretic karyotyping of typical and atypical Candida albicans.

Electrophoretic karyotypes of atypical isolates of Candida albicans, e.g., strains that were germ tube negative, failed to express proteinase activity, demonstrated low virulence for mice, formed hyperchlamydoconidia, produced hyperhyphae, or were sucrose negative (including the type strain of Candida stellatoidea), were compared with those of typical C. albicans. Karyotypes of whole-cell DNA of classical C. albicans examined with transverse alternating-field electrophoresis under specific conditions were composed of seven DNA bands with a specific migration pattern. Certain atypical strains and representatives of the three serotypes of C. stellatoidea produced discrete karyotypes with 5 to 10 bands. All isolates demonstrated a significant degree of DNA relatedness, suggesting their conspecificity. Densitometric tracings of DNA bands provided an objective and standardized method for comparing bands within the gels.

Candida albicans↗

PubMed1991

Isolation, characterization, and serial propagation of a bovine group C rotavirus in a monkey kidney cell line (MA104).

A virus (designated the Shintoku strain) which was morphologically indistinguishable from group A rotaviruses was detected in the feces of adult cows with diarrhea in Japan. The virus contained 11 segments of double-stranded RNA and had an electrophoretic migration pattern in polyacrylamide gels similar to that of other group C rotaviruses (4-3-2-2). Feces containing the bovine virus reacted with antiserum to porcine group C rotavirus (Cowden strain) but not group A or B rotaviruses in immunoelectron microscopy. The virus was adapted to serial propagation in roller tube cultures of a rhesus monkey kidney cell line (MA104) by using high concentrations of trypsin. Evidence for viral replication in MA104 cell cultures was demonstrated by immunoelectron microscopy and indirect immunofluorescence by using antiserum to porcine group C rotavirus and by electrophoretic analysis of extracted viral double-stranded RNA. A significant antibody response against the isolate was detected in convalescent-phase sera of cows which excreted the virus: no increased antibody response to bovine group A rotavirus was observed. To our knowledge, this is the first isolation of a group C rotavirus from cattle.

Animals↗

PubMed1991

Analysis of serotypes and electropherotypes of equine rotaviruses isolated in the United States.

Equine group A rotaviruses isolated over a 10-year period in New York State, New Jersey, Kentucky, and Texas were compared serotypically and electropherotypically. All isolates were determined to be serotype 3 by reaction with hyperimmune antiserum to the serotype 3 H-2 strain of equine rotavirus. All displayed RNA electrophoretic migration patterns related to that of the H-2 strain but distinct from that of serotype 5 strain H-1. A serologic survey of 184 mares in Kentucky, which was done to determine the incidence of H-1 and H-2 infections, showed geometric mean serum neutralizing titers to the H-2 strain of equine rotavirus to be significantly higher than those to the H-1 strain. These data suggest that the serotype 3 H-2 strain is the dominant equine rotavirus in Kentucky and perhaps elsewhere in the United States. We were unable to produce confirmational evidence that the H-1 strain occurs as a natural infection in the United States.

Animals↗

PubMed1995

Pathogenic, immunologic, and molecular differences between two Ehrlichia risticii strains.

Ehrlichia risticii is the causative agent of Potomac horse fever (PHF), an acute infectious disease of horses. In the last few years, there have been several reports of PHF cases occurring even in vaccinated horses. We isolated a new strain of E. risticii (90-12 strain) from a vaccinated horse suffering from clinical PHF. The major pathogenic, immunologic, and molecular differences between the 90-12 strain and the 25-D stain, which was originally isolated during the outbreaks in 1984, were studied. The 90-12 strain was more pathogenic for mice and horses compared with the 25-D strain. In enzyme-linked immunosorbent assay and immunofluorescence assay with mouse and horse antisera of both the strains, two- to fourfold differences were observed between (immunoblot) with mouse and horse antisera and also with the recombinant clone-specific antibodies. Though several antigens were similar in both the strains, there were significant differences between them in the 110-, 85-, 70-, 51-, and 33-kDa antigens. The 85-kDa antigen was present only in the 90-12 strain but cross-reacted with a 50-kDa antigen of the 25-D strain. The 51-kDa antigens of both strains had different migration patterns, Southern blot hybridization of the genome from both the strains with DNA probes made from the 51-, 55-, and clones for both the strains, whereas the probe of the 85-kDa clone showed a completely different pattern. The 16S rRNA gene sequences from the two strains were identical. Neither strain replicated in gamma interferon-treated mouse peritoneal macrophages. In in vitro neutralization assay, sera from the 25-D strain-infected horse neutralized the homologous strain but did not neutralize the 90-12 strain, whereas sera from the 90-12 strain-infected horse neutralized both the strains. In mouse protection experiments, there was complete homologous protection. But in cross-protection, mice immunized with the 25-D strain were only partially protected against challenge with the 90-12 strain, whereas mice immunized with the 90-12 strain were completely protected against the 25-D strain challenge. These results clearly indicate that there are major differences between the 90-12 and 25-D strains which may have implications regarding the vaccine failure for PHF and the development of an efficient vaccine.

Animals↗

PubMed1996

Identification of group B rotaviruses with short genome electropherotypes from adult cows with diarrhea.

Two field strains (BB-RVLV and KD) of group B rotaviruses from adult dairy cows with diarrhea displayed short genome electropherotypes. Gnotobiotic calves inoculated with fecal filtrates of each group B rotavirus developed diarrhea, and only group B rotaviruses or antigens were detected in the feces by immunoelectron microscopy and in intestinal epithelial cells by immunofluorescent staining, respectively. The feces or intestinal contents of the cows and inoculated calves were negative for group A and C rotaviruses by enzyme-linked immunosorbent assay, immunoelectron microscopy, or cell culture immunofluorescence assays. Comparison of the genome electropherotypes of the calf-passaged BB-RVLV and KD strains with the original samples and reference bovine group A, B, and C rotaviruses revealed conservative of their short-genome electropherotypes and double-stranded RNA migration patterns characteristics of group B rotaviruses. To our knowledge, our previous study (L.J. Saif, K.V. Brock, D.R. Redman, and E.M. Kohler, Vet. Rec. 128:447-449, 1991) and this report are the first description of bovine group B rotaviruses (in a mixed infection with bovine coronavirus or singly in fecal contents) in adult cows with diarrhea and this is the first report of short-genome electropherotypes among group B rotaviruses.

Animals↗

PubMed2001

Rotavirus strains bearing genotype G9 or P[9] recovered from Brazilian children with diarrhea from 1997 to 1999.

Human rotavirus strains belonging to genotype G9 or P[9] were detected in a collection of stool specimens from children with diarrhea in two cities of the state of Rio de Janeiro, Brazil, between March 1997 and December 1999. G9 strains were first detected in April 1997 and remained prevalent until the end of the study, at a frequency of 15.9% (n = 157). A high percentage of VP7 nucleotide (99.0 to 99.5%) and deduced amino acid identity (98.6 to 99.1%) was found between three randomly selected Brazilian G9 strains and the American G9 strain US1205. A novel G9:P[4] genotype combination was detected in addition to G9:P[8] and G9:P[6], demonstrating that this G genotype may undergo constant genetic reassortment in nature. The P[9] rotavirus strains constituted 10.2%, the majority of which were detected between April and July 1997. The RNA electrophoretic migration pattern of the G3:P[9] strains resembled that of AU-1 virus (G3:P3[9]), suggesting a genetic similarity between the Brazilian G3:P[9] strains and the Japanese virus, which is similar to a feline rotavirus genetically.

Antigens, Viral↗

PubMed1981

Cloning of herpes simplex virus type 1 sequences representing the whole genome.

Sequences representative of the whole genome of herpes simplex virus type 1 (HSV-1) strain KOS were cloned in the plasmid vector pBR325 in the form of EcoRI-generated DNA fragments. The cloned fragments were identified by digestion of the chimeric plasmid DNA with restriction enzymes EcoRI or EcoRI and BglII followed by comparison of their electrophoretic mobilities in agarose gels with that of similarly digested HSV-1 virion DNA. The cloned fragments showed the same migration patterns as the corresponding fragments from restricted virion DNA, indicating that no major insertions or deletions were present. The presence of HSV-1 sequences in the chimeric plasmids was confirmed by hybridization of plasmid DNA to HSV-1 virion DNA. Additionally, some of the cloned fragments were shown to be biologicaly active in that they efficiently rescued three HSV-1 temperature-sensitive mutants in cotransfection marker rescue experiments.

Cloning, Molecular↗

PubMed1982

Gene protein products of SA11 simian rotavirus genome.

When MA104 cells were infected with SA11 rotavirus, 12 protein classes, absent in mock-infected cells, could be distinguished by polyacrylamide gel electrophoresis. At least two of these proteins were glycosylated, and their synthesis could be blocked with tunicamycin. The oligosaccharides of both glycoproteins were cleaved by endo-beta-N-acetylglucosaminidase H, suggesting that they were residues of the "high-mannose" type. Of the 12 viral polypeptides observed in infected cells, 1 was probably the apoprotein of one of these glycoproteins; 5, including 1 glycoprotein, were structural components of the virions, whereas the other 6, including a second and possibly third glycoprotein, were nonstructural viral proteins. When the 11 double-stranded RNA genome segments of SA11 were translated, after denaturation, in an RNA-dependent cell-free translation system, at least 11 different polypeptides were synthesized. Ten of these polypeptides had electrophoretic migration patterns equal to those of viral proteins observed in tunicamycin-treated infected cells. Nine of the 11 double-stranded RNA genome segments were resolved by polyacrylamide gel electrophoresis and were translated individually. Two were not resolved from each other and therefore were translated together. Correlation of each synthesized polypeptide with an individual RNA segment allowed us to make a probable gene-coding assignment for the different SA11 genome segments.

Glycoproteins↗

PubMed1984

Infectious diarrhea of infant rats produced by a rotavirus-like agent.

During the investigation of an outbreak of diarrhea in suckling rats, a virus morphologically identical to but antigenically distinct from rotaviruses was identified. The disease was characterized clinically by erythema and cracking and bleeding of the perianal skin associated with the excretion of poorly formed fecal pellets, liquid, and gas. Light microscopy-observable changes consisted of small intestinal villous atrophy, villous epithelial necrosis, and villous epithelial syncytial cell formation. The cytoplasm of the epithelial syncytial cells contained large numbers of 80-nm viral particles that were often associated with reticular aggregates of electron-dense material. Viral infection principally involved the luminal one-fourth to one-third of the intestinal villi as determined by indirect immunofluorescence. This rotavirus-like agent contained 11 double-stranded RNA segments; however, the migration pattern of these segments in polyacrylamide gels differed from the electrophoretic pattern which is characteristic of the typical rotaviruses. The agent had a buoyant density in CsCl of 1.36 to 1.4 g/cm3 and was labile at pH 3 and at 56 degrees C; however, infectivity of viral inocula was not altered by extensive treatment with ether or by pH 5 buffers. This disease, which we have named infectious diarrhea of infant rats, is the first recognized viral diarrhea of rats and appears to be a good model for the study of the recently recognized group of atypical rotaviruses.

Animals↗

PubMed1985

Identification of a new polypeptide coded by reovirus gene S1.

The reovirus S1 gene has recently been shown potentially to encode two polypeptides (from two overlapping reading frames) having predicted molecular weights of 49,071 and 16,143 (Nagata et al., Nucleic Acids Res. 12:8699-8710, 1984; Bassel-Duby et al., Nature [London], in press). The larger polypeptide is reovirus protein sigma 1, but synthesis of the smaller polypeptide has not been described to date. A truncated clone of the S1 gene in which the first ATG is deleted was expressed in an in vitro protein synthesis system to yield a approximately 13-kilodalton polypeptide, as determined from migration on sodium dodecyl sulfate-polyacrylamide gels. A polypeptide with a similar migration pattern on sodium dodecyl sulfate-polyacrylamide gels was present in reovirus-infected cells and absent from mock-infected cells. Comparative tryptic peptide analysis of the 13-kilodalton polypeptides produced in vivo and in vitro showed them to be identical. Thus, the s1 mRNA of reovirus type 3 is apparently bicistronic, and we suggest that the approximately 13-kilodalton polypeptide be called sigma s (standing for sigma small).

Genes, Viral↗

PubMed1986

In vitro transcription of human pararotavirus.

Purified human pararotavirus obtained from stool samples from a 6-month-old infant was characterized. Electron microscopy of the viral particles subjected to different treatments suggested that the protein shells differed from those described for rotavirus. Treatment with both EDTA or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in the presence or absence of Mg2+ seemed to convert the virions into core particles by removal of both the outer and inner shells, and no particles equivalent to single-shelled rotavirus were observed. Different procedures were used to activate the human pararotavirus-associated RNA-dependent RNA polymerase. The enzyme was not activated by chelating agents or by thermal shock as in rotavirus. Activation by thermal shock occurred only in the presence of the four ribonucleoside triphosphates and Mg2+. However, the polymerase of pararotavirus was found to be similar to those described for rotaviruses. When in vitro transcripts were analyzed, 11 RNA species having a migration pattern similar to that of the original genomic RNA were detected.

Cell-Free System↗

PubMed1986

Regulation of expression of the wound tumor virus genome in persistently infected vector cells is related to change in translational activity of viral transcripts.

The interaction between a plant virus and its insect vector was studied at the molecular level by examining wound tumor virus (WTV) gene expression in cultured cells derived from its leafhopper vector. Infection of vector cells by WTV is noncytopathic and results in an acute phase (through day 5), followed by persistence beginning with the first cell passage. Viral-specific polypeptide synthesis and viral genome RNA accumulation increased to a maximum level during the first 5 days following inoculation and then decreased as infected cells were passaged (to 5 to 20% of the level observed during the acute phase by passages 10 to 15). In contrast, viral-specific mRNAs were present at approximately the same level in the acute phase and in the early stage (passage 10) of the persistent phase of infection. Although viral transcripts isolated at different times after inoculation exhibited identical electrophoretic migration patterns, they had different functional activities in cell-free translation systems. Transcripts isolated from persistently infected cells were inefficiently translated in vitro, reflecting the situation in infected cells. These results indicate that the decline in the level of viral polypeptide synthesis associated with the persistent phase of WTV infection is related to a change in the translational activity of viral transcripts.

Cells, Cultured↗

PubMed1990

Sequence analysis of gene 11 equivalents from "short" and "super short" strains of rotavirus.

The molecular basis for the aberrant migration pattern of the gene 11 equivalent in rotaviruses with "short" (human DS-1) and "super short" (human 69M and bovine VMRI) electropherotypes was investigated. The mRNAs of these viruses were synthesized in vitro, and the entire gene 11 equivalent of each of these viruses was sequenced with specific synthetic oligonucleotide primers. These sequences were compared with previously published sequences of "long" pattern rotavirus gene 11 segments. The increased lengths of the gene 11 equivalents of DS-1, 69M, and VMRI are due to a prolonged, 3' untranslated region in this gene segment. The 3' untranslated region of the VMRI gene 11 equivalent contains a clear duplication of a portion of its coding sequence. A stretch of 18 consecutive nucleotides within the 330-nucleotide, 3' untranslated region of 69M is identical to a section of UK coding sequence. The DS-1 and the remainder of the 69M 3'-end additional sequences are similar to each other, but neither is similar to any other currently available rotavirus gene sequence. This finding suggests that a process other than homologous duplication is involved in the evolution of these sequences. The widespread occurrence of human and animal rotaviruses with short and super short electropherotypes provides evidence that intragenic and possibly intergenic recombinational events associated with an error-prone viral RNA polymerase may play a role in increasing the genetic repertoire of rotaviruses.

Amino Acid Sequence↗

PubMed1992

Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent.

Transmissible mink encephalopathy (TME) has been transmitted to Syrian golden hamsters, and two strains of the causative agent, HYPER (HY) and DROWSY (DY), have been identified that have different biological properties. During scrapie, a TME-like disease, an endogenous cellular protein, the prion protein (PrPC), is modified (to PrPSc) and accumulates in the brain. PrPSc is partially resistant to proteases and is claimed to be an essential component of the infectious agent. Purification and analysis of PrP from hamsters infected with the HY and DY TME agent strains revealed differences in properties of PrPTME sedimentation in N-lauroylsarcosine, sensitivity to digestion with proteinase K, and migration in polyacrylamide gels. PrPC and HY PrPTME can be distinguished on the basis of their relative solubilities in detergent and protease sensitivities. PrPTME from DY-infected brain tissue shared solubility characteristics of PrP from both uninfected and HY-infected tissue. Limited protease digestion of PrPTME revealed strain-specific migration patterns upon polyacrylamide gel electrophoresis. Prolonged proteinase K treatment or N-linked deglycosylation of PrPTME did not eliminate such differences but demonstrated the PrPTME from DY-infected brain was more sensitive to protease digestion than HY PrPTME. Antigenic mapping of PrPTME with antibodies raised against synthetic peptides revealed strain-specific differences in immunoreactivity in a region of the amino-terminal end of PrPTME containing amino acid residues 89 to 103. These findings indicate that PrPTME from the two agent strains, although originating from the same host, differ in composition, conformation, or both. We conclude that PrPTME from the HY and DY strains undergo different posttranslational modifications that could explain differences in the biochemical properties of PrPTME from the two sources. Whether these strain-specific posttranslational events are directly responsible for the distinct biological properties of the HY and DY agent strains remains to be determined.

Animals↗

PubMed2005

Morphological and molecular characterization of a Cypovirus (Reoviridae) from the mosquito Uranotaenia sapphirina (Diptera: Culicidae).

A novel cypovirus has been isolated from the mosquito Uranotaenia sapphirina (UsCPV) and shown to cause a chronic infection confined to the cytoplasm of epithelial cells of the gastric ceca and posterior stomach. The production of large numbers of virions and inclusion bodies and their arrangement into paracrystalline arrays gives the gut of infected insects a distinctive blue iridescence. The virions, which were examined by electron microscopy, are icosahedral (55 to 65 nm in diameter) with a central core that is surrounded by a single capsid layer. They are usually packaged individually within cubic inclusion bodies (polyhedra, approximately 100 nm across), although two to eight virus particles were sometimes occluded together. The virus was experimentally transmitted per os to several mosquito species. The transmission rate was enhanced by the presence of magnesium ions but was inhibited by calcium ions. Most of the infected larvae survived to adulthood, and the adults retained the infection. Electrophoretic analysis of the UsCPV genome segments (using 1% agarose gels) generated a migration pattern (electropherotype) that is different from those of the 16 Cypovirus species already recognized. UsCPV genome segment 10 (Seg-10) showed no significant nucleotide sequence similarity to the corresponding segment of the other cypoviruses that have previously been analyzed, and it has different "conserved" termini. A BLAST search of the UsCPV deduced amino acid sequence also showed little similarity to Antheraea mylitta CPV-4 (67 of 290 [23%]) or Choristoneura fumiferana CPV-16 (33 of 111 [29%]). We conclude that UsCPV should be recognized as a member of a new Cypovirus species (Cypovirus 17, strain UsCPV-17).

Amino Acid Sequence↗

PubMed2006

Redox regulation of human OGG1 activity in response to cellular oxidative stress.

8-Oxoguanine (8-oxoG), a common and mutagenic form of oxidized guanine in DNA, is eliminated mainly through base excision repair. In human cells its repair is initiated by human OGG1 (hOGG1), an 8-oxoG DNA glycosylase. We investigated the effects of an acute cadmium exposure of human lymphoblastoid cells on the activity of hOGG1. We show that coinciding with alteration of the redox cellular status, the 8-oxoG DNA glycosylase activity of hOGG1 was nearly completely inhibited. However, the hOGG1 activity returned to normal levels once the redox cellular status was normalized. In vitro, the activity of purified hOGG1 was abolished by cadmium and could not be recovered by EDTA. In cells, however, the reversible inactivation of OGG1 activity by cadmium was strictly associated with reversible oxidation of the protein. Moreover, the 8-oxoG DNA glycosylase activity of purified OGG1 and that from crude extracts were modulated by cysteine-modifying agents. Oxidation of OGG1 by the thiol oxidant diamide led to inhibition of the activity and a protein migration pattern similar to that seen in cadmium-treated cells. These results suggest that cadmium inhibits hOGG1 activity mainly by indirect oxidation of critical cysteine residues and that excretion of the metal from the cells leads to normalization of the redox cell status and restoration of an active hOGG1. The results presented here unveil a novel redox-dependent mechanism for the regulation of OGG1 activity.

Cadmium↗

PubMed1993

Radiographic progression of hospital referred osteoarthritis of the hip.

Various factors have been considered important in the rate of progression of osteoarthritis of the hip, though few data are available from large longitudinal studies. One hundred and thirty six patients referred to hospital (85 women, 51 men; mean age 65 years, range 29-86 years) with osteoarthritis of the hip (192 affected hips) were followed up for a median of 27 (range 3-72) months. One hundred and six patients (144 affected hips) were reviewed for a minimum of one year (median 28, range 12-72 months). Fifty three per cent of all osteoarthritic hips required an operation at a median of 14 (range 3-48) months from entry. All radiographs before and after entry were examined (median four films for each patient over a median of three years, range 0.5-19 years). Hips showing rapid radiographic progression on prospective films more often had superior migration, or an atrophic bone response; those with no progression more often had an indeterminate, medial, or axial migration pattern, protrusio or mild osteoarthritis at presentation. A higher occurrence of rapid progression was seen in women, in those of older age at symptom onset, and in hips with a higher Kellgren grade at entry; men more often showed no progression. Age at presentation, body mass index, symptom duration, or presence of chondrocalcinosis, hand osteoarthritis/nodes, or Forestier's disease did not influence progression. This study supports the contention that, at the time of hospital referral, certain patient characteristics and radiographic features at the hip may help to identify those at high risk of rapidly progressive hip osteoarthritis.

Adult↗

PubMed1998

Identification of a common low density lipoprotein receptor mutation (C163Y) in the west of Scotland.

Familial hypercholesterolaemia (FH) is an autosomal codominant disorder characterised by high levels of LDL cholesterol and a high incidence of coronary artery disease. Our aims were to track the low density lipoprotein receptor (LDLR) gene in individual families with phenotypic FH and to identify and characterise any mutations of the LDLR gene that may be common in the west of Scotland FH population using single strand conformational polymorphism analysis (SSCP). Patient samples consisted of 80 heterozygous probands with FH, 200 subjects who were related to the probands, and a further 50 normal, unrelated control subjects. Tracking of the LDLR gene was accomplished by amplification of a 19 allele tetranucleotide microsatellite that is tightly linked to the LDLR gene locus. Primers specific for exon 4 of the LDLR gene were used to amplify genomic DNA and used for SSCP analysis. Any PCR products with different migration patterns as assessed by SSCP were then sequenced directly. In addition to identifying probands with a common mutation, family members were screened using a forced restriction site assay and analysed using microplate array diagonal gel electrophoresis (MADGE). Microsatellite D19S394 analysis was informative in 20 of 23 families studied. In these families there was no inconsistency with segregation of the FH phenotype with the LDLR locus. Of the FH probands, 15/80 had a mutant allele as assessed by SSCP using three pairs of primers covering the whole of exon 4 of the LDLR gene. Direct DNA sequencing showed that 7/15 of the probands had a C163Y mutation. Using a PCR induced restriction site assay for the enzyme RsaI and MADGE, it was determined that the C163Y mutation cosegregated with the FH phenotype in family members of the FH probands. This mutant allele was not present in any of the control subjects. Microsatellite analysis has proven useful in tracking the LDLR gene and could be used in conjunction with LDL cholesterol levels to diagnose FH, especially in children and young adults where phenotypic diagnosis can be difficult.

Adult↗