In situ hybridization for the detection of cytomegalovirus (CMV) infection

Summary Five autopsy cases of cytomegalovirus (CMV) infections were studied. Conventional light microscopy disclosed characteristic cytopathic effects in lungs, kidneys, and brain. In one case, electron microscopy was carried out and revealed typical herpesvirus particles.In situ hybridization was done with biotin-labeled CMV-DNA probes and an avidin-alkaline phosphatase detection system. 4/5 cases were observed to contain hybridizing cells in different organs. Intensity of hybridization was related to the severity of CMV infection, roughly estimated by counting cytomegalic cells. In addition to cytomegalic cells, a high number of normal-looking epithelial and mesenchymal cell types were positive. These latter cells showed nuclear hybridizations in contrast to cytomegalic cells which hybridized both within the nuclei and the cell bodies.This modified in situ hybridization procedure is a rapid and valuable tool for the detection and final demonstration of virus infection, and will be of particular help for the examination of paraffin-embedded specimens.Dedicated to Prof. Dr. G. Seifert on the occasion of his 65th birthdayThis study was supported by a grant of the Deutsche Forschungsgemeinschaft (Lo 285/2-3) and the Hamburger Stiftung zur Förderung der Krebsbekämpfung (I 208)     

In vitro infection of mononuclear cells derived from various chicken lymphoid tissues by chicken anaemia virus

Mononuclear cell suspensions derived from spleen, thymus and bone marrow tissues from chickens at 6 and 28 days of age were infected with CAV under various culture conditions. Indirect immunofluorescent staining of cytospin preparations of mononuclear cells revealed greater numbers of cells containing CAV antigens in spleen and bone marrow cultures than in thymus cultured under the same conditions. Cell cultures derived from 6- and 28-day-old chickens were equally susceptible to CAV infection. These results indicate that the age resistance to disease experimentally induced by CAV is not due to disappearance or increased resistance of a target cell. Infectivity titrations demonstrated that mononuclear cell cultures supported CAV replication. However, the virus titres obtained were lower than those detected in CAV infected MDCC-MSB1 cultures.     

Concurrent cryptosporidiosis and chicken anaemia virus infection in broiler chickens

Concurrent infection with Cryptosporidium baileyi and chicken anaemia virus (CAV) was observed in a flock of 8000 4-week-old broiler chickens. The birds, showing overt symptoms of stunted growth and 25% mortality from hatching to 4 weeks of age, harboured the protozoan in the epithelial cells of the bursa of Fabricius and the urodeal portion of the cloaca. This is the first report on an outbreak of avian cryptosporidiosis associated with CAV.     

In situ hybridization and immunohistochemistry for the detection of porcine cytomegalovirus

To establish in situ hybridization and immunohistochemistry based-assays for the detection of porcine cytomegalovirus, routinely processed renal tissue sections from 34 diseased piglets suspected of having the infection were obtained and examined. Using hematoxylin and eosin, porcine cytomegalovirus inclusion bodies were found in the nucleus of renal epithelial cells and capillary endothelial cells in the renal medulla in 30 cases. Inclusion bodies corresponding to porcine cytomegalovirus mRNA after in situ hybridization or porcine cytomegalovirus antigens after immunohistochemistry were easily determined. The cells were characterized by cytomegaly and basophilic intranuclear inclusion bodies. Using in situ hybridization, porcine cytomegalovirus mRNA were clearly detected in the nucleus and cytoplasm of the cells in 28 of the 30 (93.3%) cases. Using immunohistochemistry, porcine cytomegalovirus antigens were clearly detected in the cytoplasm of the cells in 21 of the 30 (70.0%) cases. Higher specificities and increased intensity of staining was observed with minimal background using in situ hybridization and immunohistochemistry compared with hematoxylin and eosin. Thus, the two established methods are useful and helpful tools for detecting the presence of a porcine cytomegalovirus infection.     

Detection of chicken anaemia virus by DNA hybridization and polymerase chain reaction

A clone containing the complete genome of chicken anaemia virus (CAV) was used in hybridizations with DNA from various field isolates of CAV. CAV DNA from all field isolates was detected in a polymerase chain reaction with oligonucleotides derived from the sequence of the cloned CAV DNA as primers. By way of Southern blot analysis with (32)P-labelled DNA probes derived from cloned CAV DNA, all field isolates were shown to contain DNA molecules of about 2.3 kb, i.e. the size of cloned CAV DNA. In a dot-blot assay it was demonstrated that non-radioactively-labelled cloned CAV DNA hybridized specifically to DNA from field isolates. The cloned CAV DNA is highly similar to the DNA of field isolates, as borne out by restriction-enzyme mapping. We conclude that our cloned CAV genome is representative for CAV in the field. The described PCR and hybridization techniques, may, therefore, be used for research and diagnosis of CAV infections.     

Direct detection of thermotolerant campylobacters in chicken products by PCR and in situ hybridization

We have evaluated the use of PCR and fluorescent in situ hybridization (FISH) techniques for the detection of thermotolerant campylobacters in naturally contaminated chicken products. 16S rRNA sequence data was used to design two specific primers and an oligonucleotide probe for PCR and FISH analyses, respectively. The PCR protocol amplified a 439-bp fragment corresponding to a portion of specific 16S RNA gene from thermotolerant campylobacters. The detection range of the PCR assay varied between 10 cells (after enrichment) to 10(2) cells per mL (without enrichment). FISH probes were able to identify thermotolerant Campylobacter species in 'spiked' and 'unspiked' naturally contaminated samples. PCR and FISH were performed on naturally contaminated samples and compared with the isolation of cells on selective media. The in situ hybridization technique was less sensitive than PCR, although its sensitivity of detection was increased considerably after 22 h of enrichment. These results confirm the usefulness of 16S rRNA-based techniques for the direct detection of campylobacters in food samples.     

Polymerase chain reaction amplification and in situ hybridization for the detection of human B-lymphotropic virus

Polymerase chain reaction amplification (PCR) is a recently described technique that allows for the amplification of a given sequence of DNA. It can be used to reliably amplify sequences of up to 3 kb within hours. The amplified sequence can then be recognized by hybridization with a specific probe after transfer onto nitrocellulose or nylon paper. We used PCR to recognize human B-lymphotropic virus (HBLV or HHV-6) specific sequences in various tumors as well as in the blood of patients with AIDS. Sixty-three specimens of DNA extracted from peripheral blood of patients with AIDS as well as DNA extracted from various lymphoproliferative disorders were analysed; 52 out of 63 (83%) patients with AIDS were found to have amplification of the HHV-6 specific sequence; 2 out of the 63 (3%) had equivocal amplification and 9 (14%) were found to be negative. Twenty out of 23 tumors were found to have amplified HBLV-specific sequences. Only one of these tumors was positive by Southern hybridization on restriction enzyme digested genomic DNA. In situ hybridization of clinical specimens using radiolabelled RNA probes or hapten-labelled DNA probes was used to detect the presence of HBLV in tumors. Three tumors of B cell origin were found to be positive for HBLV.     

In situ hybridization for quantitative assay of infectious hepatitis A virus.

A method of in situ hybridization using single-stranded RNA probes of opposite polarity for quantitative enumeration of hepatitis A virus (HAV) in infected cells has been developed. Kinetic experiments showed that foci of infected cells appeared as early as day 2 postinfection. The absence of foci in cells examined immediately after virus adsorption indicated that foci detected subsequently were related to viral replication. Foci were detected by hybridization with RNA probes complementary to HAV genomic RNA but not with RNA probes identical to HAV genomic RNA. The number of foci observed was linearly related to the HAV dose inoculated. Focus formation was reduced when a virus inoculum was pretreated with guinea pig anti-HAV hyperimmune serum but not when it was pretreated with preimmune serum. The high resolution of hybridization signals and relative rapidity of the test indicated that this technique will be useful for measuring serum neutralizing antibodies and for quantitative assay of infectious HAV.     

Comparison of in situ hybridization and immunohistochemistry for detection of cytomegalovirus and herpes simplex virus

In situ hybridization (ISH) and immunohistochemistry (IHC) were compared for detection of cytomegalovirus (CMV) and herpes simplex virus (HSV) in routinely processed tissue. Fifty-four formalin-fixed paraffin-embedded tissue samples infected with CMV (36 tissues) or HSV (18 tissues) from 30 autopsies were studied. All tissues had either positive viral cultures (38 of 54) or characteristic viral inclusions on hematoxylin and eosin examination (39 of 54). The tissues examined included lung (28), liver (nine), kidney (five), heart (three), adrenal (two), spleen (two), and thymus, pancreas, appendix, esophagus, and duodenum (one each). Studies by ISH were performed with two detection systems, using biotinylated probes to CMV and HSV (Enzo Biochem, New York, NY). Using ISH with an alkaline phosphatase detection system, infected cells were detected in 33 of 54 tissues (CMV: 23 of 36, HSV: 10 of 18). Using ISH with a peroxidase detection system, infected cells were identified in 30 of 54 tissues (CMV: 22 of 36, HSV: eight of 18). With IHC, antibodies to CMV and HSV stained the infected cells in 34 of 54 tissues (CMV: 24 of 36, HSV: 10 of 18). All infections detected with ISH were also detected with IHC. We conclude that these techniques for ISH and IHC are equally effective for detecting CMV and HSV in paraffin sections. The results of both techniques correlate better with viral inclusions than with culture results. The ISH stains are more difficult to prepare and in some cases are more difficult to interpret. Therefore, IHC may be preferable to ISH for detecting CMV and HSV in routine diagnostic work.     

In situ hybridization for Epstein-Barr virus NotI repeats in posttransplant lymphoproliferative disorder.

Epstein-Barr Virus-associated posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. In PTLD, B-cell expansions range from reactive hyperplasias to large cell lymphomas and are often associated with active Epstein-Barr virus (EBV) infection. The lymphoproliferations may infiltrate transplant allografts and therefore may need to be distinguished from acute cellular rejection (ACR). A total of 36 tissue specimens from 11 transplant patients (six kidney, two heart, three liver) with PTLD were studied for EBV content by automated in situ hybridization (ISH) on formalin-fixed or Bouin's-fixed, paraffin-embedded tissue using a synthetic 3' terminally biotin-labeled oligonucleotide DNA probe from the EBV NotI tandem repeat region. The NotI repeat is abundantly transcribed during productive EBV infection and may encode an EBV early antigen. EBV serologies from the 11 patients showed seven primary acute infections and one acute reactivation. Two serologic studies indicated infection of indeterminant onset, and serology was not performed on one patient. Histologically, seven patients presented with polymorphous infiltrates in transplant allograft biopsies, three of which progressed to disseminated monomorphous cell populations and death within 3 to 6 wk. Tissues examined by ISH from all 11 patients showed nuclear staining for EBV in the atypical lymphoid infiltrates (34/36 specimens). The nuclear signal ranged from a stippled pattern of positivity to homogeneous nuclear staining and was localized predominantly in follicular center cells and immunoblasts, although some smaller lymphocytes also contained the viral genome. ISHs performed on 31 allograft biopsies with ACR from 24 transplant patients (six kidney, five heart, 13 liver) without clinical evidence of PTLD and with serological evidence of past EBV infection were negative for the virus. Cell lines containing EBV in the productive state (EB3 and P3HR1) were positive with ISH for NotI, while a latently infected cell line (Raji) was negative. These data indicate that ISH with the NotI probe identifies amplified genome in EBV infections and is useful in discriminating the atypical infiltrate of EBV-associated PTLD from that seen in ACR.     

A simplified method for the detection of maedi-visna virus RNA by in situ hybridization.

A simplified in situ hybridization method for the detection of maedi-visna virus (MVV) RNA in cultured cells using 35S-labelled DNA probes is described. The protocol currently used in this laboratory for the in situ detection of MVV RNA involves paraformaldehyde fixation followed by extensive cellular pretreatment prior to hybridization. It was found that substitution of paraformaldehyde fixation with brief acetone treatment and the removal of subsequent pretreatment steps gave a similar level of hybridization signal to that of our standard protocol. Acetone fixed, non-pretreated samples were used to develop a double labelling procedure in which immunocytochemistry and in situ hybridization were combined to allow the simultaneous detection of visna virus antigens and RNA within the same cell.     

用原位酶联免疫吸附法检测甲型肝炎病毒

目的　采用一种更加简单和敏感的检测方法对甲型肝炎病毒(HAV)进行检测。方法　用甲型肝炎病毒疫苗株H2M20K株和野毒株合34感染KMB17二倍体细胞,于96孔微量细胞板上培养,并在原位用HAV特异的单克隆抗体与单层细胞上的病毒结合,辣根过氧化物酶标记的抗鼠IgG作为指示,以细胞对照A值(OD)均数的18倍判为阳性。结果　病毒增殖动态显示,高峰期为20～25d,比ELISA终点滴定法提前4～6d;两种方法同时做13个样品的感染性滴度比较,结果差异无显著意义(t=1.13,P>0.05)。原位EIA较好地指示中和试验的结果;直接从12份粪便中分离病毒,经第一代培养检测有5份阳性。结论　EIA敏感性与ELISA法相似,由于原位EIA直接检测病毒细胞系统,有操作简单,重复性好等优点,可取代ELISA终点滴定法,同时也可望用于对其他病毒细胞系统的检测。     

Comparison of in situ DNA hybridization and immunological staining with conventional virus isolation for the detection of human cytomegalovirus infection in cell cultures

The sensitivity of immunochemical staining and in situ DNA hybridization for the detection of human cytomegalovirus (HCMV) was compared with that of virus isolation. Human diploid fibroblasts were infected with serial, 10-fold dilutions of HCMV strain AD169 and examined at various intervals between 1 and 42 days after inoculation, using the three methods being compared. HCMV-DNA was detected by in situ hybridization using a biotin-labeled HCMV probe and CMV early antigen (EA) by immunochemistry using a specific monoclonal antibody. During the first 2 days after inoculation detection of EA appeared to be the most sensitive method. After the fifth day the sensitivity of the immunochemical and in situ hybridization methods was similar and equalled that of conventional virus isolation. However, 2-5 times more HCMV-DNA than HCMV-EA positive cells were detected. Our results indicate that both detection of HCMV-EA by immunological staining and HCMV-DNA by in situ hybridization are suitable methods for rapid and sensitive detection of HCMV infections.     

Identification of herpes simplex virus infection by immunoperoxidase and in situ hybridization methods

Summary Seven cases of visceral herpes simplex virus (HSV) infection were observed in five cases of hematopoietic disease and in one case each of a newborn baby and a pregnant woman. These seven cases were studied with an immunoperoxidase method and in situ hybridization. In HSV lesions of squamous epithelium, the immunoperoxidase method using rabbit anti-human HSV revealed positive staining, mainly in the nucleus but with some cytoplasmic staining. DNA in situ hybridization revealed stronger positive staining in the nucleus. In HSV hepatitis positive staining was seen in the nucleus and cytoplasm, both by immunoperoxidase and in situ hybridization methods. In the newborn baby, HSV lesions were observed in the brain only, with numerous positive astrocytes identified by the immunoperoxidase method and a few positive astrocyte nuclei by in situ hybridization. Cultured human fetal fibroblasts from the lung were infected with HSV. The immunoperoxidase method revealed diffuse positive staining in the nucleus and in the cytoplasm whereas in situ hybridization revealed fibrillar positive staining in the nucleus only. Thus, the immunoperoxidase method using rabbit antihuman HSV can detect the presence of HSV protein more sensitively than in situ hybridization, probably because of the greater quantity of HSV protein compared with HSV DNA in infected cells.     

Comparison of in situ hybridization and immunologic staining with cytopathology for detection and identification of herpes simplex virus infection in cultured cells.

Two recently developed sensitive techniques, in situ hybridization with a biotinylated cloned DNA probe and an avidin-biotin complex immunoperoxidase assay, were compared with the appearance of cytopathic changes for the early detection of herpes simplex virus infection in cell culture. By using commercially made reagents, these detection methods were evaluated in two different cell culture systems inoculated with both high- and low-input multiplicity of virus. The results revealed that both viral antigen and viral DNA detection methods could shorten the time to diagnosis of herpes simplex virus infection in cell culture; however, these methods were most useful in specimens containing low titers of virus when a less sensitive cell system was used. In this study, the avidin-biotin immunoperoxidase method was more sensitive and much cheaper than hybridization with a biotinylated probe. Significantly, when a highly sensitive cell system was used, cytopathic changes alone were comparable in rapidity and sensitivity to viral antigen or DNA detection methods applied in a less sensitive cell system.     

Immunohistochemical and in situ hybridization studies of influenza A virus infection in human lungs

Influenza viruses are responsible for acute febrile respiratory disease. When deaths occur, definitive diagnosis requires viral isolation because no characteristic viral inclusions are seen. We examined the distribution of influenza A virus in tissues from 8 patients with fatal infection using 2 immunohistochemical assays (monoclonal antibodies to nucleoprotein [NP] and hemagglutinin [HA]) and 2 in situ hybridization (ISH) assays (digoxigenin-labeled probes that hybridized to HA and NP genes). Five patients had prominent bronchitis; by immunohistochemical assay, influenza A staining was present focally in the epithelium of larger bronchi (intact and detached necrotic cells) and in rare interstitial cells. The anti-NP antibody stained primarily cell nuclei, and the anti-HA antibody stained mainly the cytoplasm. In 4 of these cases, nucleic acids (ISH) were identified in the same areas. Three patients had lymphohistiocytic alveolitis and showed no immunohistochemical or ISH staining. Both techniques were useful for detection of influenza virus antigens and nucleic acids in formalin-fixed paraffin-embedded tissues and can enable further understanding of fatal influenza A virus infections in humans.     

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.     

In situ hybridization with nonisotopic probes using different detection systems.

In order to evaluate a sensitive nonisotopic in situ hybridization method for routine work in pathology laboratories, we compared seven different detection systems, using digoxigenin- and biotin-labeled probes. The sensitivity of these methods was tested on four cases of cervical condyloma all known to be positive for HPV 6. Four of these methods gave satisfactory results without any background staining. The single biotin method and the single digoxigenin method were equally sensitive, while the two triple biotin methods, using mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase or mouse anti-biotin/alkaline phosphatase anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase as the detection systems, tremendously improved the sensitivity. The enhanced sensitivity of the nonisotopic in situ hybridization method make it useful in investigation of pathologic tissues.