[Nucleic acid based diagnosis in clinical microbiology]. / Nukleinsyrebasert diagnostikk i medisinsk mikrobiologi.

The introduction and increasing usage of nucleic acid based methods in clinical microbiology over the last years have contributed to better and earlier diagnosis of infectious diseases as well as more accurate monitoring of treatment. Various nucleic acid amplification methods such as the polymerase chain reaction and the ligase chain reaction are widely used in Norwegian clinical microbiological laboratories to detect fastidious or non-cultured infectious agents. The amplification methods combine an extremely high sensitivity with acceptable specificity. About 200,000 nucleic acid based examinations are now performed in clinical microbiological laboratories in Norway each year.

Clinical cowpox cases in Norway.

In 1994, a human and a feline case of cowpox virus infection appeared in the western part of Norway. Cowpox has not been diagnosed with certainty in Norway since the beginning of this century, when it was associated with the use of cowpox virus as a vaccine against smallpox. The human infection manifested as a spontaneously emerged, severe ulceration at the medial angle of the right eye in a 37-y-old woman, and developed into a relatively severe dermatitis. The ulcer healed slowly, leaving a scar. The feline infection was represented by a febrile, dehydrated and anorectic 6-months-old non-pedigree short-hair, with crater-like ulcers all over the body. After antibiotic and fluid therapy, revision of the skin lesions and amputation of a gangrenous toe, the cat recovered. Electron microscopy of the isolates and cultivation of virus on chorioallantoic membrane of chicken embryos confirmed the suspicion of cowpox virus infection.

Characteristics of four cowpox virus isolates from Norway and Sweden.

We report the first isolation of cowpox virus from a domestic cat in Norway, and the first confirmed isolation of cowpox virus from a human case in Norway. These two Norwegian cowpox virus isolates, as well as two Swedish human isolates, were partially characterized and compared with each other and with cowpox virus Brighton and vaccinia virus strain Western Reserve. Restriction enzyme analysis of the genomes revealed differences between all six viruses examined, but suggested that the two Norwegian isolates are closely related, as are the two Swedish isolates. Restriction endonuclease digestion of genomic DNA demonstrated that one of the Swedish isolates and the two Norwegian isolates have larger genomes than vaccinia virus strain Western Reserve, but smaller than cowpox Brighton. All four Scandinavian isolates lacked a 72 base-pair region within the A-type inclusion body protein gene which is present in the prototype cowpox virus Brighton.

[Immunoglobulin E, viral antibodies and obstructive lung disease in adults. The relation between antibody level in serum, lung function and non-specific bronchial reactivity]. / Immunglobulin E, virusantistoffer og obstruktiv lungesykdom hos voksne. Forholdet mellom antistoffnivå i serum, lungefunksjon og uspesifikk bronkial reaktivitet.

We have measured immunoglobulin E levels and respiratory virus antibodies and examined their possible role as risk factors for obstructive lung disease in adults. We observed that increased total serum IgE levels were associated with reduced lung function in subjects with obstructive lung disease, but not in asymptomatic subjects. Subjects sensitised to indoor allergens (house dust mites, cats and mould) had reduced lung function and increased, non-specific, bronchial responsiveness compared with individuals who were not sensitised to indoor allergens. Similar relationships were not observed for subjects sensitised to outdoor allergens (birch and timothy). The presence of respiratory virus antibodies was vaguely associated with reduced lung function, but was not related to increased, non-specific, bronchial responsiveness. In adults in this community sensitisation to indoor allergens is a strong predictor of reduced lung function and increased, non-specific, bronchial responsiveness, which are again closely associated with obstructive lung disease.

Histographic recording of human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev and nuclear factors.

HeLa cells and HeLa cells expressing the HIV-1 regulatory protein Rev were immunostained for Rev and pre-mRNA processing factors and examined histographically by confocal laser scanning microscopy. Following short pulse-labelling with bromouridine tri-phosphate nascent RNA gave a granular nucleoplasmic staining increasing somewhat towards the periphery as did also the heterogeneous ribonucleoproteins (hnRNPs) A1 and particularly C1/C2, a distribution pattern which has not been described. The sm-antigen of the small ribonucleoprotein particle (snRNP) proteins U1, U2, U4/U6 and U5 stained the nucleoplasm diffusely in addition to speckles which co-localised with speckles of the non-snRNP splicing factor SC-35. Brominated RNA and the hnRNPs A1 and C1/C2 were to varying degrees excluded from the speckles. Rev concentrated in the nucleolus and often as a perinucleolar ring/zone. Rev also stained the nucleoplasm and cytoplasm without co-localising with the above-mentioned proteins or brominated RNA and was not enriched or excluded in SC-35 speckles. The nucleolar proteins B23 and C23, like Rev, gave primarily a perinucleolar ring and stained the nucleoplasm but did not otherwise co-localise with Rev or with nuclear proteins. Histographic recording of immunofluorescence images proved to be a valuable tool in the study of localisation of HIV-1 Rev and cellular components and of possible co-localisations. A parallel comparison of the subcellular patterns of pre-mRNA processing factors versus major nucleolar antigens is new and suggests that the factors are not strictly separated in the nucleoplasm.

[A case of cowpox]. / Et tilfelle av kukopper.

A 37-year-old woman was admitted to the dermatology department at a regional hospital with a severe ulceration at the medial angle of the right eye. Virus culture yielded orthopoxvirus-like particles, later identified as cowpox virus. The clinical picture and virological diagnosis of cowpox are discussed. Emphasis is placed on the need for awareness among health personnel that such infections may well be encountered in an increasingly unvaccinated population. Guidelines for clinicians and for virology laboratories are given. Cats as a zoonoic source is discussed.

Oligomerization of HIV-1 Rev mutants in the cytoplasm and during nuclear import.

Oligomerization of Rev molecules has been shown to be required for Rev function. In addition to a Western blot assay monitoring dimer formation, a new in vivo assay analyzing formation of Rev heteromers in the cytoplasm and during nuclear import is presented here. The oligomerization assay is based upon the ability of Rev mutants with an intact nuclear localization signal (NLS) to interact specifically with mutants with a defective NLS and translocate such mutants to the nuclear compartments. Several of the mutants previously reported to be oligomerization defective were found to mediate nuclear and nucleolar localization of the NLS mutant. The Rev mutant previously named M4 was the only mutant tested that did not translocate the mutant with a defective NLS to the nucleus. Furthermore, the predominantly cytoplasmic localization of the M4 mutant suggests that oligomerization is important for effective nuclear import of Rev.

Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP).

Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.

HIV-1 proviral DNA sequences of env gp41 PCR amplificates from Tanzania.

The polymerase chain reaction (PCR) and direct DNA sequencing were used to detect and characterize selected regions of the HIV-1 proviral genome in whole blood samples from Tanzania. Specific PCR amplification products were obtained in gag and/or env (gp41) regions from 15 of the 19 HIV-1 seropositive samples investigated. Env regions from 12 different amplificates were further characterized using the dideoxy sequencing method. Preliminary results indicate that, despite scattered nucleotide mismatches, HIV-1 gp41 amino acid sequences from Tanzania conform to the 1990 Los Alamos African consensus sequence and resemble the HIV-1 subtype A or D consensus sequences in the characterized regions.

Serum respiratory virus antibodies: predictor of reduced one-second forced expiratory volume (FEV1) in Norwegian adults.

BACKGROUND: The purpose of this cross-sectional study was to investigate whether the presence of serum complement antibodies was associated with reduced one-second forced expiratory volume (FEV1) in adults. METHODS: From a stratified random sample of 18-73 year old adults, we performed measurements of serum complement fixing virus antibodies against influenza type A and B, parainfluenza type 1, 2, and 3, respiratory syncytial virus and adenovirus on 82% (n = 1239). RESULTS: In the crude data, subjects having five of the seven virus antibodies had significantly lower lung function, given as sex-, age- and height-standardized residuals of FEV1 (SFEV1), compared with those without. After adjusting in addition for smoking habits, lifetime smoking consumption and season, the lung function levels were significantly lower in subjects with influenza type B and respiratory syncytial virus antibodies compared to those without (P < 0.01). Increasing influenza and respiratory syncytial virus antibody titres and increasing numbers of virus antibodies, respectively, were related to progressively lower lung function. Subjects with respiratory symptoms but without obstructive lung disease had lower antibody levels than subjects with obstructive lung disease, but higher levels than asymptomatic subjects. In a final multiple linear regression analysis adjusting in addition for respiratory symptom and disease status as well as for the other respiratory virus antibodies, the presence of respiratory syncytial virus antibodies was a significant predictor for reduced SFEV1 (regression coefficient: -0.226; SE = 0.112; P = 0.04). The magnitude of the effect on lung function remained after excluding subjects reporting symptoms of respiratory infection within 3 weeks prior to the examination (regression coefficient: -0.252; SE = -0.218; P = 0.25). CONCLUSIONS: This cross-sectional community study indicates that respiratory syncytial virus infection or re-infection is an independent predictor for reduced lung function in adults of a wide age range.

Immunoglobulin G subclass antibodies to rubella virus in chronic liver disease, acute rubella and healthy controls.

Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.

[Postviral fatigue syndrome]. / Postviralt tretthetssyndrom.

The post-viral fatigue syndrome occurs sporadically and in local outbreaks (Los Angeles, Akureyri, Royal Free Hospital). The clinical picture is marked by long-lasting muscular fatigue directly following an acute infection. Other conditions associated with pronounced fatigue must be excluded. The diagnostic criteria set up by Centers for Disease Control (CDC) are the ones most commonly used. Aetiology and pathogenesis are unknown. Coxsackie B-virus seems to be associated with some cases at least. Immunological and endocrinological aberration, morphological changes in mitochondria and reduced cerebral blood perfusion have been demonstrated in some patients. There is no specific therapy. It is important for the patient that the symptoms be accepted by the doctor and society in general.

[Introduction and evaluation of HIV tests in the North-East part of Tanzania]. / Innføring og evaluering av HIV-tester i den nordøstlige del av Tanzania.

The MUTAN (Tanzanian Norwegian AIDS Project) virology programme has comprised research, intervention, surveillance and education as part of the Tanzanian National AIDS Control Programme. HIV testing of blood donors was introduced in 1988 in the regions Arusha and Kilimanjaro. Simple and rapid HIV-tests have been evaluated continuously, as well as the possible use of alternative specimen samples for testing. The polymerase chain reaction for detection of HIV proviral DNA was established at the Northern Zone Reference Hospital, The Kilimanjaro Christian Medical Centre in Moshi, in 1993.

Longitudinal study of cytomegalovirus antibodies in individuals infected with the human immunodeficiency virus.

Cytomegalovirus (CMV) antibody profiles were studied in 25 HIV-infected patients over periods of up to 56 months. Specific antibodies against CMV antigen components were monitored by complement-fixation (CF) test, EIA, Western blot and a neutralization assay. Three subjects remained CMV seronegative throughout the study. Marked fluctuations were observed in anti-CMV antibodies assayed by the CF test as compared to a control group. Fluctuations on immunoblots of purified virion antigens were also observed in the HIV-infected patients; neutralizing antibodies and anti-CMV nucleocapsid antibodies showed less variability. Seven of 22 individuals exhibited an increase in CF-test titre of up to 64-fold without clinically apparent CMV disease. On Western-blot testing of IgG reactivity with disrupted virions, ten individuals exhibited increasing reactivity to pp65, and only three of these also showed a titre rise in the CF test. In contrast, 7 of 22 showed low reactivity to the pp28 antigen. The homosexual patient group exhibited the highest levels of anti-CMV antibody. In conclusion, many asymptomatic HIV-infected subjects showed fluctuations at different levels of their antibody response to CMV, thought to be indicative of CMV reactivation/reinfection. Western-blot findings indicated that some CMV antibodies increased in level while others were lost.

Nuclear export of the human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev is mediated by its activation domain and is blocked by transdominant negative mutants.

The human immunodeficiency virus type 1 nucleocytoplasmic shuttle protein Rev moves repeatedly between the cytoplasm, a perinuclear zone, the nucleoli, and nucleoplasmic speckles. In this study, we demonstrated by both indirect immunofluorescence and Western immunoblot analysis that nuclear exit of Rev transdominant negative mutants was defective compared with that of wild-type Rev. The basic and activation domains of Rev signal import and export, respectively, of Rev across the nuclear membrane. In cotransfection experiments, mutants containing mutations of Rev inhibited the nuclear egress of wild-type Rev, thus revealing a novel transdominant negative phenotype.

Respiratory virus antibodies in adults of a Norwegian community: prevalences and risk factors.

BACKGROUND: The aims were to examine prevalences as well as demographic and environmental predictors of respiratory virus antibodies in serum. METHODS: In a cross-sectional study of 18-73 year old Norwegian adults a random stratified sample (n = 1512) was invited to attend an examination at an outpatient clinic. Seven respiratory virus antibodies were assessed by the complement fixation test. RESULTS: The attendance rate was 84%. The most frequent virus antibodies with titre of > or = 1:8 were influenza virus type A with a population standardized prevalence of 44%, adenovirus 25% and influenza virus type B 22%. The prevalences of antibodies against parainfluenza virus type 1, 2 and 3 increased with age. Smokers compared to non-smokers had an adjusted odds ratio (OR) of 1.7 (95% confidence interval [CI]: 1.3-2.4) for having one or more of the seven examined virus antibodies. The presence of one or more of the virus antibodies increased from summer to winter months (adjusted OR = 1.3 per month; 95% CI: 1.2-1.4) and it was higher in occupational dust or gas exposed smokers (adjusted OR = 2.0; 95% CI: 1.1-3.7) compared with unexposed smokers. CONCLUSIONS: Ageing, smoking, occupational dust or gas exposure as well as season of the year may thus be predictors for levels of respiratory virus antibodies in adults. These observations should be taken into account when comparing prevalences of virus antibodies in various communities as well as when examining the relationship between presence of virus antibodies and airway disease.

Raised levels of antibodies to human viruses at the clinical onset of autoimmune chronic active hepatitis.

Patients with autoimmune chronic active hepatitis (AICAH) often have very high titres of antibodies to rubella and/or measles virus. In the present study a young girl at the clinical onset of AICAH exhibited very high titres of antibodies against influenza viruses A and B, parainfluenza viruses, rubella virus and varicella-zoster virus. The titres normalized over 2 months except for rubella and varicella-zoster antibodies. Strong reactivities were seen against the rubella structural proteins E1, E2 and C in Western blot but IgM antibodies were not demonstrated. Total IgG was increased with normal ratios of subclasses. The IgG1 was the dominant antibody to E1 and E2, while IgG4 dominated the anti-C response. There was no significant shift in subclass reactivities over one year from onset. The polymerase chain reaction (PCR), using a nested primer set, was negative for rubella virus RNA in a liver biopsy obtained at the clinical onset and in peripheral blood mononuclear cells (PBMC) 1 year later. Co-cultivation experiments using PBMC and permissive cell lines were also negative for rubella virus. Hence, in the very early phase of AICAH there may be a transiently enhanced antibody response to various unrelated viruses.

The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

Elevated rubella antibodies in patients with chronic liver disease.

Patients with autoimmune chronic active hepatitis (AICAH) and certain other chronic liver disorders often have very high titres of haemagglutination-inhibition (HI) antibodies to rubella virus. In this study it is shown, using floatation centrifugation, that the high rubella HI reactivity is not caused by nonspecific lipoprotein inhibitors but rather by antibodies specific for the rubella haemagglutinin (E1 glycoprotein). After sucrose density gradient ultracentrifugation of sera the major HI reactivity was recovered in the IgG containing fractions. The IgG antibody fraction was strongly reactive by an indirect enzyme-linked immunosorbent assay (ELISA). Higher prevalence and titres of rubella antibodies were also demonstrated by the complement fixation (CF) test using a haemagglutinin-free antigen, and by an indirect haemagglutination (IHA) test (Rubacell) using a cell-associated antigen which is distinct from the antigens used in the HI and CF tests. This high rubella antibody response is therefore demonstrated using three distinct antigen-antibody systems. By means of absorption experiments and radioimmunoprecipitation assays the coating antigen used in the IHA test was shown to reside in the E2 glycoprotein. The cause of this enhanced antibody response to rubella virus structural proteins remains elusive.

Viral antibodies in infectious mononucleosis.

Patients with Epstein-Barr virus (EBV) infectious mononucleosis (IM) usually develop heterophilic antibodies and some autoantibodies. Antibodies to rubella, measles, adeno-, entero-, herpes simplex, cytomegalo- and varicella-zoster viruses were titrated in sera from IM patients and matched healthy controls using the complement fixation test (CFT) and the haemagglutination inhibition test. Except for herpes simplex virus and cytomegalovirus, the IM sera had significantly higher arithmetical and geometrical mean antibody titres and showed in most cases higher antibody prevalences in the CFT. The titre rise was most pronounced for rubella and measles antibodies, between 2- and 3-fold. There were no cases of very high titres occasionally seen in IM. The IM sera had higher total IgG serum levels than the controls, 17.27 g/l and 11.8 g/l, respectively (P < 0.001). The present data show that in addition to previously reported high levels of some autoantibodies and of heterophilic antibodies, there is a more general increase in IgG antibodies to commonly occurring viruses. This increase is most likely due to the polyclonal activation of B-lymphocytes following the binding of EBV to the complement receptor CR2 (CD21). When due consideration is given to the possible occasional occurrence of a false positive rubella IgM test, the raised antibody-titres will most likely not interfere with routine diagnostics.