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Serodiagnosis of Epstein-Barr virus (EBV) infection is currently based on the detection of antibodies to distinct EBV antigens by immunofluorescence and enzyme-linked immunosorbent assay-based tests, or in part on the detection of heterophile antibodies by the Paul-Bunnell-Davidson heterophile assay. In the past few years, the specificity and the sensitivity of serodiagnostic assay systems has been improved considerably by the use of purified recombinant EBV antigens. Screening of EBV-positive sera for antigenic reactivities by immunoprecipitation with extracts of EBV-positive cells revealed a 23-kDa protein (p23) that was recognized by antibodies from all EBV carriers tested. Open reading frame BLRF2 was identified as the coding region for this protein. After cloning and high-level expression of the BLRF2 open reading frame as DHFR fusion protein in Escherichia coli, the recombinant protein was purified to near homogeneity with the help of continuous elution electrophoresis. Sera from both EBV-positive and -negative donors were screened by immunoblot analysis and enzyme-linked immunosorbent assay for IgM and IgG antibodies against the EBV-encoded protein p23. Since anti-p23 antibodies were not detectable in 30 of 30 EBV-negative sera, and 294 of 302 EBV-positive sera had either IgM and/or IgG antibody responses to this protein, recombinant p23 seems to be a useful diagnostic marker for EBV-infection.

Genital human papillomaviruses (HPV) were detected by PCR using L1 consensus primers MY09 and MY11. To determine the underlying HPV type(s). PCR products were subsequently analyzed employing a combination of restriction fragment length polymorphism (RFLP) and hybridization with a generic oligonucleotide probe that binds to a conserved region located close to the MY11-binding site within the PCR products. Using computer-assisted sequence analysis, the lengths of the corresponding BamHI, DdeI, HaeIII, HinfI and PstI restriction fragments hybridizing with the generic probe were calculated, revealing distinct patterns for each of the 45 mucosal HPV types. This method is superior to RFLP analysis since it is not impaired by large amounts of restriction fragments resulting from nonspecific PCR products. Moreover, considering clinical specimens containing two or three different HPV types, direct sequencing of PCR products will be inconclusive, and the increased number of restriction fragments will complicate interpretation of RFLP patterns. Subsequent hybridization with the generic probe, however, results in the appearance of, at most, 2 or 3 bands per restriction enzyme digest and thus facilitates identification of the underlying HPV types.

Recent use of modern methods in the field of molecular biology has provided new insights into the genetic and structural nature of HIV, which are now increasingly being applied to the diagnostic work-up. For the reliable laboratory diagnosis of HIV infection, three different possibilities are available: detection of specific antibodies against viral proteins (anti HIV AB), detection of the virus itself (HIV antigen) and detection of viral nucleic acid using in vitro amplification techniques. In this overview, we compare clinical and diagnostic parameters of an HIV infection, and discuss the current state of the art of an HIV infection and the future prospects offered by new molecular biological methods such as the use of recombinant antigens or the polymerase chain reaction (PCR) for the detection of viral nucleic acid.

PCR-based amplification of nucleic acids has had a major impact in almost every field of basic research and has already found extensive applications in the area of clinical diagnosis. For many of these applications, quantitative data are sought to relate the quantity of amplified product to the amount of original target nucleic acid present in the sample. Since the PCR methodology with its exponential nature can be adapted for this purpose, a lot of different strategies have emerged in the last few years for sensitive and specific PCR product detection and quantification. Basic strategies, including the use of external and internal standards, are presented with respect to statistical aspects, and the advantages as well as the limitations of individual protocols are discussed. Furthermore the suitability of conventional laboratory techniques, such as gel systems or HPLC, nonradioactive labeling procedures, and the principles of advanced solid-phase-mediated strategies for the precise determination of amplification products, are outlined with the help of selected examples.

Owing to the persistence of viral DNA in leukocytes after primary CMV infection, detection of CMV DNA in these cells does not necessarily represent active infection. To identify CMV replication more precisely we have analysed immediate-early, early and late CMV transcripts by RNA amplification. The assay seems to be specific for active infection since no RNA-derived PCR products were obtained from healthy seropositive persons. The late UL83 transcript was detected in 80% of the patients with active CMV infections. Diagnosis of CMV replication by amplification of early and immediate-early transcripts was considerably less sensitive. In the case of continual CMV DNA detection in blood leukocytes by PCR without pp65 antigenemia the analysis of defined CMV transcripts would allow differentiation of active and non-active infection. In two cases the RNA assay became negative prior to DNA PCR analysis and pp65 antigen detection upon antiviral treatment, indicating that RNA amplification could be a suitable assay for early detection of the end of viral replication. No strong correlation was found between RNA detection and appearance of clinical symptoms. The development of CMV disease probably depends more on the extent of the functional impairment of the immune system.

The polymerase chain reaction (PCR) represents the most common and widespread method for the direct amplification of specific sequences of nucleic acid target molecules. Incorporation of nonradioactive labeled nucleotides during PCR by Taq DNA polymerase results in directly detectable amplification products or generates nonradioactively labeled probes for nucleic acid hybridization. Here we provide a reliable and easy to follow protocol for direct incorporation of digoxigenin-(DIG) or biotin-labeled nucleotides during PCR. The combination of high-efficient PCR amplification and high-sensitive digoxigenin technology is leading to the detection of single DNA molecules by applying digoxigenin-specific antibodies in an ELISA-type detection reaction. Following a transfer to nylon membranes, the detection of digoxigenin-labeled amplification products can also be accomplished either with a colorimetric or a chemiluminescent reaction. Using the digoxigenin-labeled amplification products as hybridization probes, sensitivities in the 0.1-pg range are obtained in Southern blot procedures.

For reproduction viruses depend on the metabolism of higher organisms that use a variety of different antiviral mechanisms for defense. Insights into virus-host cell interactions and in the strategies viruses employ to produce progeny virions were possible by sophisticated methods of modern virology. Knowing about the molecular mechanisms during infection and replication we have been able to counteract viral attacks on the molecular level by rationally designed vaccines. On the other hand these viruses can be used as molecular vector tools for treatment of e.g. genetically determined diseases. On the basis of new molecular-biological methods the sensitivity of detection of infectious agents and diagnosis of diseases could be drastically improved.

Polymerase chain reaction (PCR) was originally introduced to amplify in vitro particular DNA sequences by the application of temperature cycles (1). In a modification, RNA molecules also may serve as templates by an additional reverse transcription step converting RNA in complementary DNA sequences (2).

A new class-II restriction endonuclease, McrI, with a novel sequence specificity as isolated from the Gram-positive eubacterium Micrococcus cryophilus. McrI recognizes the palindromic hexanucleotide sequence. [sequence: see text] The novel enzyme in the presence of Mg2(+)-ions cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 3'-protruding ends with single-stranded 5'-RY-3' dinucleotide extensions. The McrI recognition sequence was deduced from mapping data on DNAs of bacteriophages theta X174RF and M13mp18RF characterized by one and four cleavage sites, respectively. The cut positions within both strands of the recognition sequence were determined in sequencing experiments by analyzing hydrolysis of phosphodiester bonds within a polylinker region of M13mp18RF DNA containing an additional McrI recognition site including treatment with T4 DNA polymerase. The novel enzyme may be a useful tool for cloning experiments by completion of the enzymes EclXI (5'-C/GGCCG-3'), NotI (5'-GC/GGCCGC-3'), PvuI (5'-CGAT/CG-3') as well as EaeI (5'-Y/GGCCR-3') and XhoII (5'-Y/GATCR-3') characterized by partly identical sequence specificities.

A noise exposure survey and audiometric assessment of 750 Los Angeles City fire fighters carried out to study the impact of fire service noise exposure on fire fighter hearing loss revealed evidence of excess hearing loss at the 3000Hz, 4000Hz, and 6000Hz test frequencies. Fire fighter medical history and life-style data did not point to a significant impact of hobbies and diseases on hearing threshold changes. The hearing loss observed at the test frequencies was, in relation to age, in excess of a general national population. This increased hearing loss with age for fire fighters suggests occupational overexposure to noise. A hearing conservation program for the fire service is therefore recommended.

A standard one-piece firefighter proximity suit (jumpsuit style) was tested for heat accumulation and hood compartment ventilation. Large increases in temperature of the skin and hood compartmental air were recorded. Oxygen and carbon dioxide were monitored and hypoxic conditions found. Using the O2 and CO2 data, mathematical regression analyses were performed to predict the time exposures allowed for firemen entering various ambient atmospheric conditions. The short permissible exposure periods predicted for the proximity suit suggests limited usefulness and the need for immediate improvements in the design of the suit.

A series of wind tunnel tests was conducted on selected fire fighter helmets to identify design factors which affect helmet ventilation at various air velocities and head orientation angles. Biomedical heat flux transducers were mounted on the surface of an electrically heated mannequin head to monitor convective heat loss. Under the experimental conditions, specific helmet design features were identified which can contribute to improved helmet ventilation and thus improve body metabolic heat loss. Attention to helmet design and helmet suspension systems is recommended to reduce fire fighter heat stress.

A pulse width tracking system is described which converts a pulse width modulated telemetry signal into an analog signal. The converter, which is based on an integrated circuit chip design, contains a feedback feature that makes it ideal for high stability servo applications. The converter was designed as an integral part of a long-range radiotelemetry system used in occupational health field research.

Occupational noise exposure was evaluated for eight fire fighter positions on board three types of emergency vehicles. One hundred seventy code-3responses were monitored and sound pressure levels in excess of 115 dBA found. Futhermore, sound pressures exceeding the OSHA allowable level of 90 dBA for eight hours exposure were determined. Octave band analyses were performed for overall code-3 noise and for specific noise sources on each vehicle. An evaluation of eighty-nine audiograms of fire fighters was also carried out. The findings of this study suggest that under present operational conditions fire fighters experience excessive short-duration, high intensity noise exposure and a hearing conservation program for the fire service is therefore recommended.

Heat stress conditions prevailing in a steel factory were evaluated using a newly developed radiotelemetry system. This system was used as a station monitor for determining environmental temperature conditions and as a personal monitor to obtain data of worker physiological responses. Environmental information was used in calculating wet-bulb globe temperature heat stress index values. Physiological data were used in confirming safety limits. The study demonstrated the effectiveness of radiotelemetry in assessing occupational exposures to heat stress.