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S Sreevatsan, J B Bookout,
F Ringpis,
V S Perumaalla,
T A Ficht,
L G Adams,
S D Hagius,
P H Elzer,
B J Bricker,
G K Kumar,
M Rajasekhar,
S Isloor,
R R Barathur
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ABSTRACT: A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.
Journal of Clinical Microbiology 08/2000; 38(7):2602-10. · 4.15 Impact Factor
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ABSTRACT: This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5' untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.
Journal of Clinical Microbiology 08/1998; 36(7):1895-901. · 4.15 Impact Factor
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ABSTRACT: Polypeptides synthesized during productive infection of HSV-1 and HSV-2 were found to possess distinct characteristics in regard to localization within the cell, DNA-binding properties, and phosphorylation after synthesis. Continuous labeling for 14 hr or pulse-labeling at successive periods during the replicative cycle with radioactive precursors revealed two types of polypeptide localization: (a) selective accumulation or enhancement within the cytoplasm or nucleus with barely detectable concentrations elsewhere and (b) accumulation in significant concentrations within both cytoplasm and nucleus showing little selective enhancement. Of the polypeptides made during HSV-1 infection 22 were phosphorylated as compared with 16 phosphoproteins specified by HSV-2. Phosphorylation was also implicated in the generation of the four molecular forms comprising the ICP 5–8 complex. Twenty-three polypeptides with affinity for DNA were detected after either type of infection. Sufficient comparisons were made to provide a basis for the tentative listing of 20 polypeptides of HSV-1 with corresponding polypeptides of HSV-2.
Virology 03/1980; 101(1):198-216. · 3.35 Impact Factor
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Virology 10/1978; 89(2):528-38. · 3.35 Impact Factor
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Virology 11/1975; 67(2):474-86. · 3.35 Impact Factor
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ABSTRACT: Serial passage of undiluted herpes simplex virus types 1 and 2 resulted in cyclic production of infectious and defective virions. Defective virus production was characterized by the appearance of a new species of viral DNA with a higher bouyant density in CsCl than standard viral DNA. Measurement of the infectivity titer and DNA synthesis revealed that the defective particles interfered with the replication of standard virions and stimulated the overproduction of a large molecular weight (175,000 daltons) polypeptide.
Intervirology 02/1975; 5(3-4):173-84. · 2.34 Impact Factor
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ABSTRACT: Mutants were isolated from γ-irradiated stocks of Schmidt-Ruppin Rous sarcoma virus (SR-RSV-2). One isolate, MI-100, displayed unusual properties in its temperature sensitivity of cell-transforming capabilities. Focus formation, colony formation in soft agar, and increased [3H]deoxyglucose uptake by infected cells were all rendered temperature sensitive (ts); 37° was permissive for expression of these functions while 41° was nonpermissive. Tumorigenicity in chickens was greatly diminished. Virus replication was not defective since virus yields exceeded those of wild-type RSV at 37 and 41°. Yields of infectious progeny may have been higher than shown by titrations, as virions of MI-100 were more heat labile than wild type. Group-specific antigen concentrations in MI-100 infected were almost equivalent at 37 and 41° and greater at both temperatures than those of wild-type-infected cells.Through genetic recombination with Rous-associated virus (RAV-1), subgroup specificity of MI-100 was altered from B to A without correcting the temperature sensitivity of transformation. However, temperature-shift experiments demonstrated a major difference in the ts properties of the mutant and its recombinant. MI-100 was unable to transform cells at 37° if initial incubation after infection was at 41° (irreversible inhibition); also, shifts of infected cells, at any time, from 37 to 41° resulted in loss of the transformed phenotype (reversible inhibition). Cells infected with the recombinant still required permissive temperature in order to express the transformed phenotype, but initial incubation at 41° after infection did not affect the ability to transform cells at 37°. Thus there appeared to be ts lesions in MI-100 affecting transformation, one in an early function and one related to a late function. Recombination with RAV-1 restored the early but not the late functions, suggesting that leukosis viruses possess a gene coding for some initial event in fibroblast transformation but lack the gene(s) required for full expression (maintenance) of the transformed state. Moreover, as shown by the replicative properties of the mutant, the early gene of transformation initiation is apparently not involved in virus replication.
Virology.
