Role of respiratory viral infection in sids: Detection of viral nucleic acid by in situ hybridization

There is considerable evidence suggesting that respiratory viral infection is involved in the genesis of the sudden infant death syndrome (SIDS), with rates of about 20 per cent of SIDS victims compared to about 13 per cent of controls. Since the techniques used previously are prone to under-reporting from autopsy material, non-isotopic in situ hybridization (NISH) has been used to detect viral nucleic acid in lung in SIDS. Forty-five SIDS cases (30 males) were examined (age range 3 weeks–14 months, mean age 3·9 months). Thirty non-SIDS cases (15 males) were also examined (age range 5 weeks–24 months, mean age 9·0 months). Eleven of 45 (24·4 per cent) SIDS cases were positive by NISH compared to 1 of 30 (3·3 per cent) non-SIDS cases (P=0·012). There were eight cases of adenovirus type 5, two cases of respiratory syncytial virus (RSV), and one case of parainfluenza virus type 2. The one positive control case was adenovirus type 5. Only lung parenchyma was examined here. Additional examination of the upper respiratory tract may increase the number of positive cases.     

Cellular localization of cytomegalovirus nucleic acids in guinea pig salivary glands by in situ hybridization

In situ hybridization with guinea pig cytomegalovirus (CMV) DNA probes was used to detect guinea pig CMV nucleic acid sequences in salivary glands during the course of infection. Optimum conditions for detection of guinea pig CMV gene sequences at the cellular level were determined. The technique of nucleic acid hybridization was compared to antigen detection and histologic examination for cellular localization of guinea pig CMV during acute and chronic infection. Tissue sections fixed for 1 h in 4% paraformaldehyde demonstrated darker specific staining and contained significantly larger numbers of cells positive for guinea pig CMV nucleic acids and antigens than sections fixed longer and in other fixatives. The method of in situ hybridization detected more guinea pig CMV infected cells than did routine histological evaluation. Histologically normal salivary gland duct cells as well as cells bearing typical inclusions were found to contain guinea pig CMV nucleic acids. Guinea pig CMV nucleic acids were also detected although less frequently in cells outside of the ducts. These results suggest that in situ hybridization allows for the detection of histologically inapparent guinea pig CMV infections at the cellular level.     

In situ hybridization studies for viral nucleic acids in heart and lung allograft biopsies

In situ hybridization has been shown to be a useful technique for the identification of specific viruses in pathologic tissues. The authors studied 313 lung and 164 heart biopsies from 20 heart-lung recipients to assess its utility in this clinical setting, employing biotinylated probes for the cytomegalovirus, herpes simplex, and adenovirus genomes. Twenty-five lung biopsies and one heart biopsy had detectable cytomegalovirus DNA by in situ hybridization. As compared to histopathology, in situ hybridization had a sensitivity of 85% and a specificity of 99%. None of the biopsies had detectable herpes simplex or adenovirus by either in situ hybridization or routine histopathology. In situ hybridization studies may be of greatest use when the results of conventional histopathology are equivocal and in the patients with radiologic or clinical evidence of pulmonary disease.     

Ultrastructural localization of human papilloma virus by nonradioactive in situ hybridization on tissue of human cervical intraepithelial neoplasia.

BACKGROUND: A nonradioactive in situ hybridization was developed to localize human papilloma virus (HPV) at the ultrastructural level. EXPERIMENTAL DESIGN: Cervical biopsies from human uterine cervices clinically suspicious of condyloma were embedded in Lowicryl K4M at low temperature. Postembedding in situ hybridization was performed with DNA probes specific for HPV types 6/11, 16, and 18. The hybrids were detected by anti-horseradish peroxidase antibodies conjugated with 10 nm colloidal gold particles. RESULTS: Localization for HPV 16 and 18 both was to intranuclear and cytoplasmic sites. Cytoplasmic detected HPV signals were between masses of intermediate filaments and in vacuoles; other organelles were devoid of positive signal. Within the nucleus the precise localization of the viral nucleic acid was episomal, vacuolar, and chromosomal. In situ hybridization with plasmid control DNA confirmed the specificity of the HPV positive signals. CONCLUSIONS: This study helps define the subcellular compartmentalization of HPV DNA in infected human cells.     

Direct detection and identification of African trypanosomes by fluorescence in situ hybridization with peptide nucleic acid probes

We have developed a rapid and easy to perform fluorescence in situ hybridization test that allows specific identification of trypanosomes from the subgenus Trypanozoon, using peptide nucleic acid probes. Probes were designed to target subgenus-specific sequences on the multiple-copy 18S rRNA, greatly facilitating the detection of a single trypanosome.     

Identification of Fusarium species in formalin-fixed and paraffin-embedded sections by in situ hybridization using peptide nucleic acid probes

Fusarium has recently emerged as an opportunistic pathogen of humans, but the histological differentiation of Fusarium from Aspergillus and Scedosporium is particularly difficult because these fungi may induce similar clinical features and exhibit filamentous development in host tissues. Thus, there is a need to establish rapid and reliable methods that are applicable to pathological diagnoses. The aim of this study was to evaluate and establish in situ hybridization (ISH) using peptide nucleic acid (PNA) probes targeting the 28S rRNA to identify Fusarium species in tissue sections. This technique was validated using both formalin-fixed and paraffin-embedded pulmonary tissues from mice infected with seven different species of fungi and cell blocks from fungal cultures of 30 strains. As a result, strong positive signals were observed within fungal organisms present in tissues of the lung from mice infected with Fusarium solani. Furthermore, this probe reacted strongly with both F. solani and Fusarium oxysporum in sections from cell blocks. Although some cross-reactivity occurred with the Pseudallescheria boydii in sections from cell blocks, the signal intensity was low and most hyphae were not reactive. In conclusion, it was confirmed that ISH with PNA probes is accurate and is a valuable tool for identifying Fusarium spp. among organisms that have identical morphological features in formalin-fixed and paraffin-embedded sections.     

Diagnosis of fetal rubella infection by nucleic acid hybridization

The efficacy of nucleic acid hybridization for the diagnosis of rubella infection in experimental and clinical materials was compared with immunoblot and virus isolation techniques. Our results showed that nucleic acid hybridization is specific and rapid but gives false-negative results when compared with conventional virus isolation in some experimental although not in clinical materials so far examined. For this reason, a failure to demonstrate rubella virus in fetal specimens by this method alone cannot yet be taken as a sole criterion for ruling out fetal rubella infection.     

Chromosomal localization of three humanras genes by in situ molecular hybridization

Three human rasfamily protooncogenes, c-Ki-ras-1, and c-Ki-ras-2, and N-ras,have been mapped to chromosome bands 6p11–12, 12p11.1–12.1, and 1p11–13, respectively by in situ molecular hybridization. Certain human cancers display consistent and specific alterations involving chromosomes 1, 6, and 12. The precise chromosomal localization of ras genes will permit evaluation of thepossible effect of these chromosome changes on the structure and activities of ras protooncogenes in human neoplasia.     

Detection of adenoviruses in stool specimens by nucleic acid spot hybridization

Nucleic acid hybridization was used for the detection of adenovirus DNA in stool specimens, and the results were compared with those obtained by a radioimmunoassay (RIA) for adenovirus hexon antigen. DNA from 40 specimens, 18 of which were positive by RIA, were spotted onto nitrocellulose filters and analyzed by hybridization using radioactively labeled adenovirus-2 DNA or a cloned DNA fragment from enteric adenovirus-41 as probes. With the adenovirus-2 DNA probe, 15 of the 18 RIA-positive specimens were also positive in the hybridization assay, and one of the RIA negative specimens was also scored as positive. The cloned adenovirus-41 fragment gave a positive signal with five specimens, all of which were also detected with the adenovirus-2 DNA probe. The results show that hybridization is an alternative method for detection of adenovirus in stool specimens. The sensitivity of the assay is comparable to that of the RIA.     

Comparison of reporter molecules for viral in situ hybridization

A number of streptavidin-linked reporter molecules at the endpoint of a five-step detection protocol for viral in situ hybridization using biotinylated probes were examined. DNA-DNA and RNA-RNA model systems were used. Streptavidin linked to either peroxidase or fluorescein was found to be optimal in terms of sensitivity and resolution within individual cells. All other reporter molecules labelled similar numbers of cells with low background reaction. However, streptavidin-5 nm gold followed by silver enhancement gave very high background staining making interpretation of positive signals very difficult.     

Rapid identification of bluetongue virus by nucleic acid hybridization in solution

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with ³⁵S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 μl reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.     

用核酸分子杂交技术产前诊断人巨细胞病毒感染

用ELISA法筛选早孕妇女和中孕引产妇女积压人巨细胞病毒IgM(HCMV-IgM)，早孕妇女阳性者在旱12．20周(中孕引产妇女阳性者在引产时)留羊水，用生物素标记的HCMV-DNA探针斑点杂吏检测其羊水中的HCMV-DNA。对胎儿巨细胞病毒惑染作出产前诊断。结果：早孕妇女384例．血HCMV-IgM阳性26例，此26例羊水HCMV-DNA阳性9例，其中7例出生时留脐血进一步检测HCMV-IgM和HCMV-DNA。证实有HCMV感染。中孕引产妇女125例。血HCMV-IgM阳性10例。此10例羊水HCMV-DNA阳性4例。     

In vitro detection of canine distemper virus nucleic acid with a virus-specific cDNA probe by dot-blot and in situ hybridization

A cDNA library was prepared from canine distemper viral (CDV) messenger RNA (mRNA) derived from Vero cells lytically infected with the Onderstepoort strain (Ond) of CDV. A 300 base pair insert was identified which, by Northern blot analysis and Sanger sequence data, was shown to be specific to the nucleocapsid gene. The nucleocapsid (NC) clone was radiolabelled with 32P using nick translation and used to detect viral RNA in both dot-blot and in situ preparations of Vero cells lytically infected with Onderstepoort CDV (Ond-CDV) and immortalized mink lung cells persistently infected with racoon origin CDV (CCL64-RCDV). Dot-blot hybridization results paralleled immunofluorescent results in the lytically infected cells. In 18 persistently infected cell lines from the RCDV-CCL64 parental stock, 13 lines were positive and two were negative on both immunofluorescence and dot-blot hybridization analysis for CDV antigen and RNA, respectively. Viral nucleic acid was detected in these persistently infected cells, where as few as 1.9% of the members of a line were positive on immunofluorescence. A dot-blot autoradiographic signal was obtained in three lines which were negative for CDV antigen. CDV RNA was detected in both lytically and persistently infected cell lines by in situ hybridization, where decreasing probe length was important in increasing the sensitivity of this assay. Viral RNA was detected in over 90% of the lytically infected cells, where only 70% were positive for viral antigen by immunofluorescence.     

Detection of Fiji disease virus in infected sugarcane by nucleic acid hybridization

Clones of Fiji disease virus were obtained by cDNA synthesis from isolated viral genomic double-stranded RNA. Nick-translated probes made from a mixture of clones specifically detected Fiji disease virus in infected sugarcane galls, leaves and growing tips. This method for detection of Fiji disease virus is extremely sensitive, and can be used as a rapid screening procedure for the virus with very small tissue samples.