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PubMed2005

Reverse transcriptase-polymerase chain reaction to detect avian encephalomyelitis virus.

A reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and optimized for the detection of avian encephalomyelitis virus (AEV). A pair of primers was prepared based on the VP2 gene of the structural protein P1 region of the AEV genome. An avian encephalomyelitis virus-specific 619-base pair cDNA product was amplified by these primers from five reference/field strains of AEVs but not from 10 other avian pathogenic viruses and bacteria. The RT-PCR assay developed in this study was found to be sensitive and specific with as little as 10 pg of avian encephalomyelitis virus RNA detected using gel electrophoresis. Furthermore, AEV-RT-PCR was able to detect AE virus from chicken embryo brain at 3 days postinoculation as compared with the AE agar gel precipitation test (AGP), which required up to 11 days of incubation in the embryos.

Animals↗
PubMed1979

Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.

Animals↗
PubMed1987

Altered structure and expression of c-myc in feline T-cell tumours.

The c-myc gene is rearranged in a subset of feline T-cell lymphosarcomas. Detailed mapping of c-myc rearrangements showed that some result from feline leukaemia virus (FeLV) proviral integration within or upstream of c-myc, but one case involves a complex 3' alteration and amplification which is apparently not directly virus-induced. S1 nuclease mapping of RNA from normal cells using c-myc probes revealed two presumptive 5' ends, each corresponding to a promoter-like sequence (P1 and P2), and a major 3' discontinuity which mapped to the 3'-most of two possible polyadenylation signals. Analysis of RNA from a series of tumours revealed different modes of c-myc expression. All tumours produced P1 and P2 transcripts with apparently normal structure except for one case where an insertion in intron 1 displaced exon 1 sequences. The abundance ratio of P1/P2 transcripts varied considerably and was high in tumours which carry a rearrangement adjacent to c-myc, but some other T-cell tumours with no apparent myc alteration displayed an equally high ratio. However, a consistent feature was the lack of detectable RNA from normal c-myc alleles in tumours which express a rearranged c-myc allele or a transduced FeLV v-myc gene. We suggest that this may prove to be a useful indicator of the presence of an oncogenically active myc gene, whether this is a rearranged c-myc or transduced v-myc sequence.

Alleles↗
PubMed1998

Molecular identification and functional characterization of a novel protein that mediates the attachment of erythroblasts to macrophages.

We have previously identified a novel protein that mediates the attachment of erythroblasts to macrophages in vitro. This attachment promotes terminal maturation and enucleation of erythroblasts (Hanspal and Hanspal, Blood 84:3494, 1994). This protein is referred to here as Emp for erythroblast macrophage protein. Two immunologically related isoforms of Emp with apparent molecular weights of 33 kD and 36 kD were detected in macrophage membranes. The complete amino acid sequence of the larger isoform of Emp was deduced from the nucleotide sequence of a full-length 2.0-kb cDNA that was isolated from a human macrophage cDNA library using affinity-purified anti-Emp antibodies. Of the 2,005 bp, 1,185 bp encode for 395 amino acids representing 43 kD (the sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] molecular mass is 36 kD). Northern blot analysis of human macrophage poly(A) RNA detected a message for Emp of 2.1 kb. The deduced amino acid sequence contains a putative transmembrane domain near the N-terminus. To investigate the structure/function relationships of Emp, recombinant fusion proteins of full-length and truncated Emp were produced in bacteria, COS-7, and HeLa cells. Cell binding assays showed that the N-terminus is exposed on the cell surface. The recombinant Emp functions as a cell attachment molecule when expressed in heterologous cells. Furthermore, we showed that the demise of erythroblasts in the absence of Emp-mediated erythroblast-macrophage association is accompanied by apoptosis. We postulate that Emp-mediated contact between erythroblasts and macrophages promotes terminal maturation of erythroid cells by suppressing apoptosis.

Amino Acid Sequence↗
PubMed2003

Detection of feline coronavirus infection in southern African nondomestic felids.

Feline coronavirus (FCoV) infects members of the Felidae family with results ranging from seroconversion with no disease to fatal feline infectious peritonitis (FIP). Infection of non-domestic felids with FCoV is of concern, particularly in endangered populations such as cheetahs (Acinonyx jubatus). In this investigation, we tested 342 animals in the Republic of South Africa and Namibia, including 140 animals from wild populations, for evidence of FCoV infection by serology and/or reverse transcription/nested polymerase chain reaction (RT/nPCR) on feces from 1999 through 2001. Past or current infection was evaluated. Of these, 195 animals had evidence of infection and included 41 animals from wild populations. Serology (indirect immunofluorescence) did not always correlate with viral RNA detection, as seronegative animals were occasionally virus-positive, while many seropositive animals were not shedding virus. Serology indicated the infecting virus was most closely related to type I FCoV. Antibody levels in the majority of animals were low, even in those actively infected. Ten of 48 animals tested at more than one time point by RT/nPCR were shedding virus at multiple time points possibly indicating persistent infection. Infection in free-ranging animals was also notable, as over a quarter of the free-ranging animals tested had evidence of current or previous FCoV infection. Testing by serology and RT/nPCR is recommended for screening for FCoV infection.

Africa, Southern↗
PubMed2000

Quantitation of human immunodeficiency virus type 1 RNA in different biological compartments.

Little information is available describing viral loads in body fluids other than blood. In addition, the suitability of commercially available assays for human immunodeficiency virus type 1 (HIV-1) RNA quantitation has not been evaluated in most nonblood fluids. We compared Organon Teknika's nucleic acid sequence-based amplification method (NASBA) and Roche's Amplicor HIV-1 Monitor (reverse transcriptase PCR [RT-PCR]) for quantitating HIV-1 RNA in cerebrospinal fluid (CSF), saliva, breast milk, seminal plasma, and cervical-vaginal lavage fluid (CVL). Saliva and breast milk frequently demonstrated some inhibition in the RT-PCR assay, similar to the inhibition previously described in seminal plasma. Inhibition of the RT-PCR assay was not observed with CSF or CVL, nor in any of the NASBA assays. When fluids from HIV-infected individuals were tested by RT-PCR and NASBA, 73 and 27% of CSF samples and 60 and 40% of breast milk specimens had detectable RNA, respectively. These differences were not statistically significant. In cross-sectional studies using RT-PCR to measure viral RNA in paired blood plasma and CSF samples, 71% of blood plasma samples and 42% of CSF samples were positive. A similar analysis using NASBA with paired blood plasma and CVL, saliva, or seminal plasma samples revealed 91% were blood plasma positive and 55% were CVL positive, 76% were blood plasma positive and 46% were saliva positive, and 83% were blood plasma positive and 63% were seminal plasma positive. NASBA worked fairly well to quantitate HIV-1 RNA from all fluids without apparent inhibition. RT-PCR performed well on CVL and CSF, frequently with greater sensitivity, although its use in other fluids appears limited due to the presence of inhibitors. These studies demonstrate that viral loads in nonblood fluids were generally lower than in blood.

Body Fluids↗
PubMed2002

Whole stimulated salivary flow in patients with chronic hepatitis C virus infection.

BACKGROUND: Salivary gland disorders have been included among the extra-hepatic manifestations related to HCV infection. METHODS: The whole stimulated salivary flow rate (spitting technique) was studied in 74 HCV infected patients to evaluate salivary gland dysfunction. RESULTS: The salivary flow of the patients with chronic HCV infection was similar to that of the healthy controls. The association between subjective xerostomia salivary flow was seen to be very weak. No significant associations were found between salivary flow and age, sex, risk factor of acquired infection, ALT, AST, GGT, ALP values, time lapsed since the diagnosis or HCV-RNA detection in saliva. CONCLUSIONS: Although the functional repercussion of hepatitis C related lymphocytic sialoadenitis remains unclear, we did not find a significant reduction in the whole stimulated salivary flow in HCV infected patients.

Adult↗
PubMed2003

Latent hepatitis B virus (HBV) infection and HBV DNA integration is associated with further transformation of hepatoma cells in vitro.

Hepatitis B virus (HBV) is the major cause of chronic liver disease and hepatocellular carcinoma in the world, with more than 400 million people infected worldwide. To date, there is no reliable model for the study of the many aspects of HBV infection, despite the use of the chimpanzee. Although several alternative methods have been previously developed for the in vitro study of HBV infection, there is still an urgent need for new in vitro infection models, including for the ability of HBV to integrate into the host cell genome. Here we describe a process to improve infection of the human hepatoma cell lines HepG2 and HuH-7 in vitro with HBV originating from human blood. As shown previously for infection of hepatocytes with hepatitis C virus (HCV), the removal of the cell-bound lipoproteins prior to the addition of the viral inoculum to the cells could also be critical for the uptake of HBV via lipoprotein (LDL)-related receptors. Induction by insulin and dexamethasone led to an increase of HBsAg expression at the cell surface in association with the integration of the viral DNA into the host genome and HBx RNA detection. This integration process was also shown to be associated with cytopathic changes and further phenotypic transformations of the cells.

Animal Testing Alternatives↗
PubMed2003

Hepatitis C virus: prevalence and routes of infection among blood donors of West Mexico.

In Latin America few studies have explored frequency and risk factors predicting hepatitis C virus (HCV) infection in blood donors. In this study we determined the prevalence of HCV infection in blood donors from West Mexico. Potential risk factors, clinical, histological and virologic characteristics presented in this group were also evaluated. METHODS: HCV antibodies were evaluated in 57108 blood donors with commercial second-generation enzyme immunoassays. Positive results were confirmed by a recombinant immunoblot assay. Repeatedly seropositive donors were further studied for risk factors, history for hepatitis, hepatic enzymes (aspartate aminotransferase and alanine aminotransferase (AST and ALT)), liver histology and hepatitis C virus-ribonucleic acid (HCV-RNA) detection. RESULTS: A total of 499 blood donors were initially tested positive doubtful for antibodies to HCV Ab (0.8%). While there was no difference in HCV prevalence with respect to age or gender, the most frequent risk factors identified were transfusion (42%), household exposure (14.8%), multiple sex partner (6.8%) and intranasal cocaine use (2.3%). Also, we found that from a subgroup of donors tested for histological analysis, 19% presented abnormal ALT levels and 90% showed abnormal liver histology. No correlation was found between abnormal ALT levels and the presence of HCV-RNA in serum. CONCLUSION: These results demonstrate a low prevalence (0.8%) of HCV infection among Western Mexican blood donors, which was comparable to those established for Western countries, but in contrast in our study the most frequent risk factor continues being transfusion followed by household exposure.

Journal Article↗
PubMed2003

Alternatives to HIV-1 RNA concentration and CD4 count to predict mortality in HIV-1-infected children in resource-poor settings.

Cheaper, simpler alternatives to CD4 lymphocyte count and HIV-1 RNA detection for assessing the prognosis of HIV-1 infection are needed for resource-poor settings. However, little is known about the predictive value of alternative assays, in particular in children. We assessed the prognostic value of total lymphocyte count, immune complex-dissociated p24 antigen, white blood cell count, packed-cell volume (haematocrit), and serum albumin for mortality in 376 HIV-1-infected, mainly African-American or Hispanic children enrolled during March, 1988 to January, 1991. In a Cox proportional hazards model, including all assay-alternatives to CD4 and RNA, total lymphocyte count (p<0.0001) and serum albumin (p=0.0107) independently predicted mortality. Further assessment of these markers is warranted in resource-poor settings.

Biomarkers↗
PubMed2005

Universal linkers for signal amplification in auto-ligating probes.

We reported recently oligonucleotide ligation methods for detection of DNAs and RNAs in solution and in cellular imaging. In previous systems, ligated full-length oligonucleotide products have almost native DNA structure and bind tightly with target strand, which limits the resulting signals to one per target. When small amounts of RNAs are targeted, signal amplification becomes very important issue. Here, we report on a new universal linker to destabilize ligated products in template-dependent auto-ligation, which accelerates the dissociation of ligated product from target and allows as much as 92-fold amplification of signals in DNA and RNA detection without enzymes, reagents, or thermal cycling. This signal amplification is shown in solution experiments and in solid supported assays.

DNA↗
PubMed1993

Ultrastructural distribution of DNA and RNA within the nucleolus of human Sertoli cells as seen by molecular immunocytochemistry.

The precise distribution of DNA and RNA within the human Sertoli cell nucleolus has been investigated, at the ultrastructural level, by cytochemical and molecular immunocytochemical techniques. In Sertoli cells, the nucleolar components show a typical spatial distribution. The fibrillar centres are not surrounded by a layer of dense fibrillar component, but come in contact only with strands of dense fibrillar component. These fibrillar parts of strands are the extensions of granular strands connected to a large granular mass. These strands delimit numerous nucleolar interstices in which chromatin fibres are clearly obvious. Using the in situ terminal deoxynucleotidyl transferase/immunogold procedure for detecting DNA, we find evident label exclusively over the chromatin fibres enclosed in the nucleolar interstices and over the fibrillar centres, and no significant label over the dense fibrillar component and granular component of the nucleolus. Furthermore, using the polyadenylate nucleotidyl transferase/immunogold procedure for detecting RNA, we show that label is deposited not only over the granular component and dense fibrillar component, as expected, but also quite obviously over the fibrillar centres. No label is seen over the interstices containing chromatin.

Cell Nucleolus↗
PubMed1996

Newer aspects of pathogenesis of heart failure: hepatitis C virus infection in myocarditis and cardiomyopathy.

Myocarditis is thought to be commonly caused by various viruses, and accumulating evidence links viral myocarditis with the eventual development of dilated cardiomyopathy. In many cases, however, the evidence is only circumstantial, and direct conclusive proof is not available. Polymerase chain reaction (PCR) has been used to detect enterovirus RNA in myocardial tissue, but the wide discrepancy in results emphasizes the need for further study. We evaluated patients with cardiomyopathy and myocarditis by PCR for the presence of various RNA and DNA viruses in the heart. Hepatitis C virus RNA was found in high frequency, but enterovirus RNA was detected only in low frequency. Therefore, we investigated hepatitis C virus infection in patients with dilated cardiomyopathy. Antihepatitis C virus antibody was present in the sera of 6 of 36 patients (16.7%) with dilated cardiomyopathy and in 1 of 40 patients (2.5%) with ischemic heart disease, showing a statistically significant (P < .05) difference. Hepatitis C virus RNA was present in the sera of four of the six patients, and all four had hepatitis C virus type II. The copy number of hepatitis C virus RNA in the serum was 8 x 10(2) to 2 x 10(3) genomes per 1 mL serum. Positive strands of hepatitis C virus were found in the hearts of three patients, and negative strands of hepatitis C virus were detected in the hearts of two patients. The results suggest that hepatitis C virus infection is frequently found in patients with dilated cardiomyopathy and that hepatitis C virus is an important causal agent in the pathogenesis of the disease. Antiviral therapy against hepatitis C virus may be indicated in these patients.

Adult↗
PubMed1992

Mouse dopamine beta-hydroxylase: primary structure deduced from the cDNA sequence and exon/intron organization of the gene.

Genomic clones for mouse dopamine beta-hydroxylase (DBH) were isolated from two genomic libraries derived from DBA/2J and 129/SV mouse strains, by plaque hybridization with the human DBH cDNA probe. Subsequently, cDNA encoding mouse DBH was amplified with reverse transcription-polymerase chain reaction (RT-PCR) method using primers corresponding to 5'- and 3'-portions of the mouse DBH mRNA, subcloned into a plasmid vector, and subjected to nucleotide sequence analysis. The clone encoded a protein of 621 amino acids with a calculated molecular mass of 70,186 daltons. The predicted amino acid sequence of mouse DBH showed 87%, 80% and 79% identities with the rat, bovine and human enzymes, respectively. Several potential amino acid sequences that are involved in the posttranslational modification and catalytic function of DBH were identified in mouse DBH protein. Nucleotide sequence analysis of the overlapping genomic clones showed that the mouse DBH gene was composed of 12 exons about 17 kb in length. Typical TATA and CCAAT boxes were observed in the 5'-upstream region of the gene. Northern blot analysis of adrenal gland RNA detected a single size species of the mouse DBH mRNA.

Adrenal Glands↗
PubMed2006

Avian hepatitis E virus in an outbreak of hepatitis--splenomegaly syndrome and fatty liver haemorrhage syndrome in two flaxseed-fed layer flocks in Ontario.

Two commercial layer chicken flocks that were fed a flax-based diet beginning at 28 weeks of age for the production of omega-3 fatty-acid-enriched eggs experienced increased mortality when the birds reached 37 weeks. The average weekly mortality was 0.34% over a 20-week period, with peak mortality of 0.9% for 1 week. Reduced feed consumption, reduced body weight gain and poor peak production were noticed prior to the onset of increased mortality. A total of 245 birds were necropsied and 78% of these had lesions in the liver and spleen, with 44% of those necropsied having changes consistent with hepatitis-splenomegaly syndrome, with lesions ranging from acute periportal lymphoplasmacytic hepatitis to chronic severe cholangiohepatitis with haemorrhage, vasculitis and amyloidosis. A total of 11% of the birds had lesions typical of fatty liver haemorrhagic syndrome, and 22% had lesions found in both hepatitis-splenomegaly syndrome and fatty liver haemorrhagic syndrome. No significant bacteria or viruses were recovered from samples of the liver/bile or spleen but 11 of 21 bile samples contained avian hepatitis E virus RNA detectable with a reverse transcriptase-polymerase chain reaction assay. Comparative sequence analysis found identities of 82 to 92% and 78 to 80% between the helicase and capsid protein genes, respectively, of the virus detected in this outbreak and those of other avian hepatitis E virus isolates, suggesting extensive genetic heterogeneity in avian hepatitis E viruses in Ontario flocks.

Animal Feed↗
PubMed1998

Epizootic hemorrhagic disease: analysis of tissues by amplification and in situ hybridization reveals widespread orbivirus infection at low copy numbers.

A recent outbreak of hemorrhagic fever in wild ruminants in the northwest United States was characterized by rapid onset of fever, followed shortly thereafter by hemorrhage and death. As a result, a confirmed 1,000 white-tailed deer and pronghorn antelope died over the course of 3 months. Lesions were multisystemic and included severe edema, congestion, acute vascular necrosis, and hemorrhage. Animals that died with clinical signs and/or lesions consistent with hemorrhagic fever had antibody to epizootic hemorrhagic disease virus serotype 2 (EHDV-2) by radioimmune precipitation but the antibody was limited exclusively to class immunoglobulin M. These findings, indicative of acute infection, were corroborated by the observation that numerous deer were found dead; however, clinically affected deer were rarely seen during the outbreak. Furthermore, only in animals with hemorrhagic lesions was EHDV-2 isolated and/or erythrocyte-associated EHDV-2 RNA detected by serotype-specific reverse transcription (RT)-PCR. By using a novel RT in situ PCR assay, viral nucleic acid was localized to the cytoplasm of large numbers of tissue leukocytes and vascular endothelium in tissues with hemorrhage and to vessels, demonstrating acute intimal and medial necrosis. Because PCR amplification prior to in situ hybridization was essential for detecting EHDV, the virus copy number within individual cells was low, <20 virus copies. These findings suggest that massive covert infection characterized by rapid dissemination of virus facilitates the severe and lethal nature of this disease.

Animals↗
PubMed2001

Hepatitis C prevalence and risk factors in hemodialysis patients in Central Brazil: a survey by polymerase chain reaction and serological methods.

An hemodialysis population in Central Brazil was screened by polymerase chain reaction (PCR) and serological methods to assess the prevalence of hepatitis C virus (HCV) infection and to investigate associated risk factors. All hemodialysis patients (n=428) were interviewed in eight dialysis units in Goiânia city. Blood samples were collected and serum samples screened for anti-HCV antibodies by an enzyme-linked immunosorbent assay (ELISA). Positive samples were retested for confirmation with a line immunoassay (LIA). All samples were also tested for HCV RNA by the PCR. An overall prevalence of 46.7% (CI 95%: 42-51.5) was found, ranging from 20.7% (CI 95%: 8.8-38.1) to 90.4% (CI 95%: 79.9-96.4) depending on the dialysis unit. Of the 428 patients, 185 were found to be seropositive by ELISA, and 167 were confirmed positive by LIA, resulting in an anti-HCV prevalence of 39%. A total of 131 patients were HCV RNA-positive. HCV viremia was present in 63.5% of the anti-HCV-positive patients and in 10.3% of the anti-HCV-negative patients. Univariate analysis of risk factors showed that the number of previous blood transfusions, transfusion of blood before mandatory screening for anti-HCV, length of time on hemodialysis, and treatment in multiple units were associated with HCV positivity. However, multivariate analysis revealed that blood transfusion before screening for anti-HCV and length of time on hemodialysis were significantly associated with HCV infection in this population. These data suggest that nosocomial transmission may play a role in the spread of HCV in the dialysis units studied. In addition to anti-HCV screening, HCV RNA detection is necessary for the diagnosis of HCV infection in hemodialysis patients.

Adolescent↗
PubMed1997

Expression of nitric oxide synthases and GTP cyclohydrolase I in the ventilatory and limb muscles during endotoxemia.

Nitric oxide synthases (NOS) convert L-arginine to nitric oxide in the presence of tetrahydrobiopterin (BH4). Two constitutive isoforms of NOS exist in normal skeletal muscle fibers, however, the existence of a third, the inducible isoform (iNOS), has never been detected in these fibers in vivo. Therefore, we assessed the influence of in vivo endotoxemia on skeletal muscle expression of constitutive and inducible NOS isoforms and GTP cyclohydrolase I, the rate limiting enzyme of BH4 synthesis. Two groups of rats were infused i.p. either with E. coli endotoxin (20 mg/kg, LPS group) or saline (saline group). Animals were killed 6 h later and the ventilatory and limb muscles were quickly frozen. Endotoxin infusion elicited a significant rise in NOS activity of the diaphragm, intercostal, soleus and gastrocnemius muscles. Reverse transcription-polymerase chain reaction (RT-PCR) on muscle total RNA detected very low expression of iNOS and GTP cyclohydrolase I mRNA in the saline group, but significant upregulation of both enzymes was found in the ventilatory and limb muscles of the LPS group. Immunoblotting detected no iNOS protein in the saline group but a significant iNOS protein expression was found in the diaphragm, intercostal and soleus muscles and to a lesser extent, in the gastrocnemius of the LPS group. Endotoxemia was also associated with increased protein expression of constitutive NOS isoforms mainly in the diaphragm and to lesser extent in the intercostal, gastrocnemius and soleus muscles. We conclude that in vivo exposure to endotoxin leads to differential induction of both iNOS and GTP cyclohydrolase I in the ventilatory and limb muscles.

Animals↗