In Situ Polymerase Chain Reaction Research Articles

Biological functions of endophytic bacteria in Robinia pseudoacacia 'Hongsen'.

Endophytes and their host plants have co-evolved for a very long time. This relationship has led to the general recognition of endophytes as a particular class of microbial resources. R. pseudoacacia 'Hongsen' is drought- and barren-resistant species that can be grown in both the north and south of China, efficiently addresses the ecological issues caused by China's 'southern eucalyptus and northern poplar. Up to date, cultured-dependent studies are available for the R. pseudoacacia nitrogen-fixing and other endophytes. Therefore, the present research studied the R. pseudoacacia 'Hongsen,' microbiome in detail by high-throughput sequencing and culture dependant. This study examined microbial species and functional diversity in Robinia pseudoacacia 'Hongsen' using culture-dependent (isolation) and culture-independent techniques. A total of 210 isolates were isolated from R. pseudoacacia 'Hongsen.' These isolates were clustered into 16 groups by the In Situ PCR (IS-PCR) fingerprinting patterns. 16S rRNA gene sequence analysis of the representative strain of each group revealed that these groups belonged to 16 species of 8 genera, demonstrating the diversity of endophytes in R. pseudoacacia 'Hongsen'. 'Bacillus is the most prevalent genus among all the endophytic bacteria. High-throughput sequencing of endophytic bacteria from R. pseudoacacia 'Hongsen' of the plant and the rhizosphere soil bacteria showed that the bacterial populations of soil near the root, leaf, and rhizosphere differed significantly. The microbial abundance decreased in the endophytes as compared to the rhizosphere. We observed a similar community structure of roots and leaves. With and without root nodules, Mesorhizobium sp. was significantly different in R. pseudoacacia 'Hongsen' plant. It was predicted that R. pseudoacacia 'Hongsen' plant endophytic bacteria would play a significant role in the metabolic process, such as carbohydrate metabolism, amino acid metabolism, membrane transport, and energy metabolism.

Abstract B30: Bovine leukemia virus in breast tissue linked to increased cell proliferation and breast cancer risk

Abstract Purpose: To compare recent data sets on frequency of bovine leukemia virus (BLV) detection in breast tissue in different human populations, and association of BLV with a confirmed diagnosis of breast cancer and expression of common clinical biomarkers. Many risk factors are associated with breast cancer, but what causes initial molecular/cellular changes from normal to malignant is not well understood. Six types of human cancer are caused by viruses, and several viruses have been studied for a role in breast cancer. BLV is a common virus of US cattle (38% of beef herds, 89% of dairy herds, 90-100% of large dairy operations). BLV DNA and protein were previously identified in human tissues. Methods: Breast tissue specimens were archived formalin-fixed, paraffin-embedded tissue sections from sources mentioned below. In situ PCR (IS-PCR) was performed on intact deparaffinized sections on glass slides, using primers from the tax region (transforming gene), highly conserved and rarely deleted, as are most BLV genome regions as tumor progression occurs. BLAST sequence comparisons indicated primers had high homology only with BLV, and extremely low homology with human genomic sequences, including human endogenous retroviral sequences. Previous laboratory experiments confirmed this specificity and established appropriate controls for the assay. Sections were semiquantitatively scored for density of the digoxygenin-labeled IS-PCR product within mammary epithelial cells, upon microscopic examination by two independent examiners. Results: Previous data sets using samples from the Cooperative Human Tissue Network, USA (n=239) and New South Wales, Australia (n=96), showed BLV presence significantly more frequently in malignant tissue than in normal breast tissue from women with no breast cancer history. Odds ratios (OR) and confidence intervals (CI) were 3.07(1.66-5.69), p≤.001 and 4.72(1.71-13.05), p≤.003, respectively. New unpublished data indicate specimens from MD Anderson Cancer Center in Houston, TX (n=216) show an even higher OR (CI) of 5.59 (2.76-11.30), p≤.0001. Argentinian specimens showed lower frequency (22%) in malignant tissues than US (58%) and Australian specimens (80%). Cell proliferation markers, KI67 and HER2 overexpression were significantly associated with BLV presence in Argentinian malignant breast tissues. For 31 Australian subjects with breast cancer, archived normal breast tissues were available from surgery 3-10 years previous for an unrelated condition. For 23 (74%) of these, BLV was already present in normal tissue at least 3 years before cancer diagnosis, consistent with a causative temporal relationship between BLV presence and subsequent cancer development. Conclusions: New unpublished data indicate BLV in some Argentinian women, but the frequency in breast cancer tissue is lower than in US and Australian women. Tests for conventional prognosis/proliferation biomarkers (ER, PR, HER2, Ki67) in Argentinian specimens showed BLV presence significantly associated with moderate-high Ki67 level (p≤0.009) and HER2 overexpression (p≤0.0442), consistent with possible BLV role in enhanced cell proliferation. BLV’s mode of transformation is via the oncogenic protein (Tax), which inhibits host cell DNA repair via the base excision pathway. DNA damage observed in breast cancer cells, including driver mutations, could theoretically have been initiated due to DNA repair inhibition by BLV infection. Elucidating initiators for any cancer opens opportunities for primary and secondary prevention. For viruses, the reservoir (e.g., BLV-infected cattle) could be reduced, transmission to humans could be intercepted (e.g., education to avoid raw milk, raw beef appetizers), vaccines could be developed, and elimination of the virus might be achieved by developing BLV-targeting agents. Driver mutations already initiated by BLV might be targeted as part of personalized secondary prevention and/or therapy. Note: This abstract was not presented at the conference. Citation Format: Gertrude C. Buehring, HuaMIn Shen, Kimberly Baltzell, Jennette Sison, Savitri Krishnamurty, Pamela Lendez, Lucia Matinez-Cuesta, MariaVictoria Nieto-Farias, GuillerminaLaura Dolcini, MariaCarolina Ceriani. Bovine leukemia virus in breast tissue linked to increased cell proliferation and breast cancer risk [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B30.

Investigating porcine parvoviruses genogroup 2 infection using in situ polymerase chain reaction

BackgroundPorcine parvovirus 2 (PPV2) was detected in swine serum without showing any relationship with disease. The emergence of the virus seemed to be a unique event until other genetically highly similar parvoviruses were identified in China and, later in 2012, the presence of the virus was also described in Europe. PPV2 is widely distributed in pig populations where it is suspected to be involved in respiratory conditions, based on its frequent detection in lung samples. In order to investigate the potential pathogenic involvement of PPV2, 60 dead pigs were examined from two farms. They were necropsied and tested for PPV2 and PCV2 (Porcine circovirus type 2) by PCR; by Brown and Brenn (B&B) staining for bacteria; by immunohistochemistry (IHC) to detect CD3, Swine leukocyte antigen class II DQ (SLAIIDQ), lysozyme, porcine reproductive and respiratory syndrome virus (PRRSV), swine influenza (SIV), Mycoplasma hyopneumoniae (Mhyo); and by in situ hybridization (ISH) to detect ssDNA and dsDNA of PCV2. PPV2 positive samples were subjected to in situ polymerase chain reaction (IS-PCR) including double staining method to detect PPV2 and host cell markers. To calculate statistical difference we used GENMOD or LOGISTIC procedures in Statistical Analysis System (SAS®).ResultsWe found that the PPV2 was localized mostly in lymphocytes in lungs, lymph nodes and liver. Neither CD3 antigen nor lysozyme was expressed by these infected cells. In contrast, low levels of SLAIIDQ were expressed by infected cells, suggesting that PPV2 may have a specific tropism for immature B lymphocytes and/or NK lymphocytes though possibly not T lymphocytes.ConclusionThe overall conclusion of this study indicates that PPV2 may contribute to the pathogenesis of pneumonia.

Application of in situ PCR for the Detection of Bovine Leukaemia Virus (BLV) Infection in Dendritic Cell cultures

Abstract The aim of the study was to develop an in situ PCR (IS-PCR) method for detection of bovine leukemia virus (BLV) in cell cultures. Samples from five BLV positive and five BLV negative cows were collected and dendritic cells (DCs) from blood, bone marrow, spleen, and lymph node were cultured. Cultures prepared from healthy animals were infected with BLV. After two weeks, the cells were tested by nested PCR and IS-PCR for the presence of proviral DNA. As a positive control adherent cell line permanently infected with BLV was used. BLV was successfully detected by IS-PCR in DCs from naturally infected cattle and DCs infected in vitro. In control, non-infected DCs, the results of the reaction were negative. The results of provirus detection by IS-PCR were similar with these performed with nested PCR. Additionally, IS-PCR provides many advantages, like specific localisation of infection and smaller number of cells needed as template for PCR.

Nanobarcoding: detecting nanoparticles in biological samples using in situ polymerase chain reaction.

BackgroundDetermination of the fate of nanoparticles (NPs) in a biological system, or NP biodistribution, is critical in evaluating an NP formulation for nanomedicine. Current methods to determine NP biodistribution are greatly inadequate, due to their limited detection thresholds. Herein, proof of concept of a novel method for improved NP detection based on in situ polymerase chain reaction (ISPCR), coined “nanobarcoding,” is demonstrated.MethodsNanobarcoded superparamagnetic iron oxide nanoparticles (NB-SPIONs) were characterized by dynamic light scattering, zeta potential, and hyperspectral imaging measurements. Cellular uptake of Cy5-labeled NB-SPIONs (Cy5-NB-SPIONs) was imaged by confocal microscopy. The feasibility of the nanobarcoding method was first validated by solution-phase PCR and “pseudo”-ISPCR before implementation in the model in vitro system of HeLa human cervical adenocarcinoma cells, a cell line commonly used for ISPCR-mediated detection of human papilloma virus (HPV).ResultsDynamic light-scattering measurements showed that NB conjugation stabilized SPION size in different dispersion media compared to that of its precursor, carboxylated SPIONs (COOH-SPIONs), while the zeta potential became more positive after NB conjugation. Hyperspectral imaging confirmed NB conjugation and showed that the NB completely covered the SPION surface. Solution-phase PCR and pseudo-ISPCR showed that the expected amplicons were exclusively generated from the NB-SPIONs in a dose-dependent manner. Although confocal microscopy revealed minimal cellular uptake of Cy5-NB-SPIONs at 50 nM over 24 hours in individual cells, ISPCR detected definitive NB-SPION signals inside HeLa cells over large sample areas.ConclusionProof of concept of the nanobarcoding method has been demonstrated in in vitro systems, but the technique needs further development before its widespread use as a standardized assay.

Epstein–Barr virus is seldom found in mammary epithelium of breast cancer tissue using in situ molecular methods

Epstein-Barr virus (EBV) has been proposed as a possible etiological agent of breast cancer based on 21 reports of EBV in malignant breast tissues. Most of these studies used standard and nested solution polymerase chain reaction (PCR) techniques, both disadvantaged by susceptibility to contamination from laboratory EBV, and the inability to localize the signal to a specific cell type. To avoid these issues, we used in situ molecular methods of viral detection to reassess the frequency of EBV in malignant breast tissue. We used a commercial in situ hybridization (ISH) system with an EBER genome target, and a non-commercial in situ PCR (IS-PCR) method using primers specific for the BamH1 region. The assays were performed on malignant breast tissue sections from 70 breast cancer patients at the M. D. Anderson Cancer Center, Houston, TX. EBV was found in mammary epithelial cells, the cell type from which most breast cancers arise, in 2/70 (2.9%) of specimens using IS-PCR and in none of the specimens using ISH. Based on these findings that EBV was present in human mammary epithelial cells so infrequently, it is unlikely to play a causative role in most types of breast cancer.

Limited evidence of human papillomavirus on breast tissue using molecular in situ methods

Human papillomavirus (HPV) has been proposed as an etiologic agent of breast cancer based on numerous reports of high-risk (oncogenic) HPV types in malignant breast tissues. However, most of those studies used standard and nested solution polymerase chain reaction (PCR) techniques, both of which are disadvantaged by vulnerability to laboratory contamination from positive control DNA and the inability to localize the signal to a specific cell type. To overcome these drawbacks, the authors of this report explored the use of in situ molecular methods of viral detection to reassess the frequency of HPV in malignant breast tissue. In situ hybridization (ISH) was used with probes that were specific for the capsid region of 12 oncogenic HPV types, and in situ PCR (IS-PCR) was used with primers that were specific for the capsid region of HPV-16, which is the most common oncogenic HPV type. These methods were resistant to molecular contamination and allowed identification of the positive cell type. The specimens examined were malignant tissues from patients with 70 breast cancer patients at The University of Texas M. D. Anderson Cancer Center in Houston, Texas. HPV was observed in 4 of 70 specimens (5.7%) using ISH and in 2 of 70 specimens (2.9%) of specimens using IS-PCR. Concordance between the 2 methods was high for negative specimens; both methods yielded negative results in 66 of 70 specimens (94.3%). However, there was no concordance for the few positive specimens, probably because of differences in sensitivity and the targeted HPV types. Oncogenic (high-risk) HPV types were present in malignant breast epithelium very infrequently and, thus, may be causative agents of only a relatively small proportion of all breast cancers.

A novel in situ polymerase chain reaction hybridisation assay for the direct detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded tissues

An in situ polymerase chain reaction (IS-PCR) hybridisation assay was carried out on the brains of 20 cattle infected naturally with bovine herpesvirus type 5 (BoHV-5). Sections from the olfactory bulb and the frontal cortex of each sample were analysed using IS-PCR followed by hybridisation targeting the BoHV-5 US9 gene using a biotinylated primer. Each of the IS-PCR and hybridisation steps was optimised, and three different methods for detecting the virus were used. No false positive signals were observed in any negative control sample ( n = 20), resulting in a specificity of 100%. The results of IS-PCR hybridisation analysis of the olfactory bulb and the frontal cortex be compared directly with the results obtained using virus isolation, and the specificity and sensitivity were calculated. The most suitable method of visualisation was the peroxidase/3′-3-diaminobenzidine (DAB) detection system coupled with the use of the fluorescent dye Cy3. Using either of these methods, 80% of the positive samples (16 out of 20 samples) were identified using olfactory bulb sections. This is the first report using IS-PCR hybridisation for the direct detection of BoHV-5 DNA in clinical samples, and it provides an additional method for veterinary virology.

Detection of Epstein Barr virus in formalin-fixed paraffin tissues by fluorescent direct in situ PCR

Specific viral laboratory diagnosis of primary Epstein-Barr Virus (EBV) infection is usually based on antibody-detection assays. However, molecular detection is also considered the reference standard assay for diagnosis of central nervous system infections and of most cases of nasopharyngeal carcinoma (NPC). One-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus-specific Ig antibodies for the laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis. In this article, we will present a rapid, sensitive and robust molecular tool for the viral detection of EBV (EBNA-1) within tissue specimens by making use of in situ PCR (IS-PCR).

Detection of Haemophilus influenzae type B DNA in a murine pneumonia model by in situ PCR

This study estimated the value of in situ PCR (ISPCR) in the detection of Haemophilus influenzae type b (Hib) DNA in paraffin-embedded lung tissues of a murine pneumonia model. ICR mice were infected with Hib solution intranasally. In study group A (n=20), physiological changes and the number of deaths were recorded for 7 consecutive days after infection. In study group B (n=10), blood samples and lung tissues were obtained from the infected mice on the brink of death. In both groups, portions of the lung tissue were cultured for Hib, while other portions were submitted for histopathological studies. Conventional PCR, PCR followed by Southern blotting and ISPCR were performed to detect Hib in paraffin-embedded lung tissues. In control group A, six mice were inoculated intranasally with the same concentration of heat-inactivated Hib solution. In control group B, six healthy mice served as a blank control. Both control groups were managed using the same methods as those used in the study groups. The white blood cell count of the mice in the study group increased (F=3.295, P<0.01), with a high neutrophil count (F=0.127, P<0.05). In the histopathological study, various stages of pneumonia were found in the lung tissues of the infected mice examined by microscope; 80% of the mice had moderate or severe pneumonia. Cultures of lung tissues in the study groups were all positive for Hib, while no bacteria were found in the control groups. Hib was detected in only 4 of 30 samples (13.3%) of the study groups using conventional PCR, but in all 30 samples (100%) using both Southern blotting and ISPCR. All three methods did not detect Hib in the control groups. Because of its sensitivity and specificity and its ability to locate the micro-organism, ISPCR can be considered suitable for the detection of Hib in paraffin-embedded lung tissues.

Detection of human papillomavirus type 16 DNA in formalin-fixed, paraffin-embedded tissue specimens of gastric carcinoma

Human papillomavirus type 16 (HPV16) is regarded as one of the important tumor-related viruses, which are known to have a role in cervical carcinoma; however, there are few reports on HPV16 in gastric carcinoma (GC). Our study aimed to investigate the relationship between HPV16 and the occurrence of GC. Liquid PCR (LPCR) and in-situ PCR (ISPCR) methods were carried out to detect the HPV16 oncogene E6 cell-type-specific enhancer in the long control region of HPV16 in 40 GCs and corresponding gastric adjacent normal mucosa (GANM). The patients were from Shaanxi Province in China; Helicobacter pylori (Hp) was detected by immunohistochemistry and by hematoxylin and eosin staining in their gastric tissues. The HPV16 E6 gene was detected in 37.5% (15/40) of the GCs and 5% (2/40) of the GANMs with LPCR, as was the cell-type-specific enhancer; however, the positive rate of E6 was 27.5% (11/40) in GCs and 0% (0/40) in GANMs, respectively, with ISPCR. HPV16 DNA was mainly located in the nucleus of gastric glandular epithelium cells. The infection rate of HPV16 DNA in GCs was higher than that in GANMs (P=0.0004), and the HPV16 had no statistical correlations with sex, age, invasion, grading or lymph node metastasis (P>0.05). The infection rate of HPV16 in cardiac GCs was significantly higher than that in noncardiac ones (P=0.0136), and HPV16 had no correlation with Hp in GCs (P=0.0829). Receiver operator characteristic curve analysis indicated that there was no statistical difference between the LPCR and ISPCR methods in our study through optimizing parameters in ISPCR procedures (P=0.768). These findings suggested that HPV16 can infect gastric glandular epithelium cells and that viral infection might play a role in the occurrence of GCs independent of or without the cooperation of an Hp infection.

The pattern of viral persistence in monkeys intra-tracheally infected with Simian varicella virus

In situ PCR (ISPCR) and in situ hybridisation (ISH) was performed on 32 tissues from 10 monkeys, intra-tracheally (IT) infected with simian varicella virus (SVV) and 5 tissues from 3 uninfected control animals. The results showed persistence of SVV DNA up to 2 years post-infection (pi) and the localisation of SVV to be confined to neurons except at time points 9 and 10 months pi where SVV positive satellite cells were also detected. There was no evidence for transcription of SVV ORFs 63 and 21 in the ganglia of the one IT infected and 2 naturally infected monkeys investigated using RNA ISH.

Primary synovial sarcoma of the kidney: corroboration with in situ polymerase chain reaction

Synovial sarcoma is a tumor of the soft tissues with a unique chromosomal translocation t(X;18)(p 11.2;q11.2) that can be detected by polymerase chain reaction in tissue homogenates. The case of a 32-year-old woman with a primary synovial sarcoma of the kidney is described, the diagnosis was corroborated by the recently developed method of in situ polymerase chain reaction (IS-PCR). Synovial sarcoma of the kidney may be confused with other spindle cell tumors, for that reason IS-PCR may be useful to confirm the diagnosis in paraffin-embedded material.

Detection of Rabies Virus Genes by In-Situ Polymerase Chain Reaction

Rabies is diagnosed by FAT in the impression smears of brain tissues. In this study, an attempt was made to diagnose rabies using in situ polymerase chain reaction (ISPCR). A digoxigenin-labelled double-stranded probe specific for a portion of the 'N' gene of rabies virus was used. Positive signals were identified as blue dots in the intraneuronal and neuropil areas.

Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction.

The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.

Detection of human immunodeficiency virus-1 RNA and DNA by extractive and in situ PCR in unprocessed semen and seminal fractions isolated by semen-washing procedure

To determine the presence of human immunodeficiency virus-1 (HIV-1) viral RNA/DNA in whole semen, in properly isolated seminal fractions and in spermatozoa after swim-up, by extractive nested PCR and to compare the detection of HIV DNA by in situ PCR (IS-PCR) with the results of nested PCR. We tested HIV-1 RNA and DNA by nested PCR in semen and in seminal fractions from 55 patients. Non-spermatic cells and spermatozoa pellet fractions from 10 HIV-1-positive and five HIV-1-negative men were tested for proviral DNA by IS-PCR. All samples of spermatozoa recovered after sperm washing were free of HIV RNA. HIV RNA tested positive in seven (13%) seminal plasma samples and only in two (4.2%) whole semen of these same samples. Of the seven seminal plasma samples testing positive for HIV RNA, four men had elevated blood viral load and three an undetectable viraemia. HIV DNA by IS-PCR turned positive in three of five samples in semen of HIV-noninfected men. HIV RNA/DNA detection in the semen of HIV-infected men proves the efficacy of sperm washing with swim-up of spermatozoa. It is recommended that nested PCR be conducted on purified seminal compartments. IS-PCR is inadequate for detecting HIV in semen.

Locatization of human herpesvirus type 8 in human sperms by in situ PCR

Defining the mechanism of infection with human herpesvirus-8 (HHV-8) or Kaposi's sarcoma associated herpesvirus (KSHV) is an important clinical issue. HHV-8 has been linked to Kaposi's sarcoma (KS) development in HIV-1-infected individuals, and KS develops in 40% of those infected with both viruses. A series of epidemiological data suggest that sexual transmission is one of the routes of transmission for HHV-8. In our studies, we sought to assess the cellular reservoirs of HHV-8 DNA in the semen of HIV-1-infected men and the potential role of HHV-8 infected spermatozoa in horizontal transmission. A nested polymerase chain reaction (PCR), in situ PCR (ISPCR) and a sodium iodide (NaI) DNA isolation technique that extracts both nuclear and episomal DNA were utilized to amplify specific genes in vitro and within intact cells to evaluate the types of seminal cells infected with HHV-8 in HIV-1-infected and uninfected men. HHV-8 was present in the spermatozoa and mononuclear cells of the semen in 64 of 73 (88%) HIV-1 infected individuals. Both the sperms as well as the mononuclear cells of the semen specimens of HIV-1 infected men were found to be infected with HHV-8. Multiplex ISPCR revealed that a significantly higher percentage of semen cells were infected with HHV-8 than HIV-1 (p>0.001). Rare (less than one in a 100,000) sperm cells were co-infected with both viruses. A co-culture of HHV-8 infected sperm with uninfected 293 or Sup-T1 cell lines resulted in an abortive infection of these cells with HHV-8. DNA isolation by NaI yielded 73% of the positive sperm, whereas the standard phenol/chloroform method resulted in significantly lower positives (45%) from the same specimens. Design and methods: Our data strongly suggest a potential sexual/horizontal route of transmission of HHV-8, via the HHV-8 infected sperm and other semen cells, where a large percentage of HIV-1 infected men's sperm and other semen cells are infected with HHV-8. Co-culture studies have further supported the observations that HHV-8 in the sperm cells is infectious and capable of transmission of the virus to uninfected cells.

Highly Sensitive Detection and Localization of Maternally Acquired Human Cytomegalovirus in Placental Tissue by In Situ Polymerase Chain Reaction

Transplacental transmission of human cytomegalovirus (CMV) can result in congenital malformations, although details on the mechanisms of transmission and the location of CMV in infected placentae need to be described.METHODS. Placental tissue from term (third trimester) deliveries was screened for CMV infection by polymerase chain reaction (PCR), in situ PCR (IS-PCR), and IS reverse-transcriptase PCR (IS RT-PCR).RESULTS. CMV DNA was detected in tissue samples from 11 placentae that had been determined to be negative for CMV during routine pathological examination. IS-PCR demonstrated the presence of CMV DNA in all cell types within placental villi, and IS RT-PCR further defined this result by identifying viral transcripts from all stages of replication. CMV DNA and RNA were shown to be highly concentrated in placental trophoblast cells. The infecting viruses were detected with primers specific for the major immediate early section of the genome (UL122/123), the UL21.5 virion gene, and the glycoprotein B (gB) gene and were determined to be predominantly genotype gB2. Therefore, maternal and fetal host factors, as well as viral load and possibly viral genotype, may all affect the outcome of placental CMV infection.CONCLUSION. Placental villi are involved in the transfer of blood from maternal to fetal circulation. Infection and replication of CMV within placental trophoblasts suggests that these structures may be involved in the transmission of CMV.

Comparison of a novel in situ polymerase chain reaction (ISPCR) method to other methods for white spot syndrome virus (WSSV) detection in Penaeus vannamei

Penaeus vannamei were experimentally injected with white spot syndrome virus (WSSV) and tested for WSSV at different times post-injection (p.i.) by 1-step polymerase chain reaction (PCR), 2-step PCR, in situ hybridization (ISH) and in situ polymerase chain reaction (ISPCR) in order to compare sensitivity of the methods. With 1-step PCR, 4 of 15 shrimp tested positive for WSSV at 12 h p.i., and all tested positive by 24 h p.i. With 2-step PCR, 13 out of 15 samples tested positive at 2 h p.i. and all were positive by 4 h p.i. Using in situ hybridization, 1 sample tested positive at 18 h p.i. and all were positive by 36 h p.i. With ISPCR, 1 out of 5 samples was positive at 2 h p.i. and all were positive by 8 h p.i. Two-step PCR showed the highest sensitivity, followed by ISPCR, 1-step PCR and ISH. Although ISPCR revealed WSSV in 9 of 10 P. vannamei that tested positive for WSSV using 2-step PCR, none of the shrimp examined showed clinical signs of WSSV infection or detectable WSSV with 1-step PCR. The major infected organs were muscle and the hepatopancreas.

Prolonged cold preservation promotes the recipient's cell participating in neointima formation but delays the later graft arteriosclerosis in rat model

Chronic graft dysfunction is the greatest barrier to long-term graft survival, although the immediate outcome in organ transplantation has been greatly improved. Graft arteriosclerosis is a prominent feature of chronic graft dysfunction. Recipient progenitor cells have been shown to participate in neointimal development in graft arteriosclerosis. The present study investigated the role of recipient endothelial cells in the repair and remodeling after a cold preservation injury in an orthotopic cross-sex abdominal aortic allotransplantation model, namely female Wistar to male Sprague-Dawley rats. Grafts were preserved for 48 hours in 4°C University of Wisconsin (UW) solution for a prolonged cold ischemia (PCI) group or preserved for <1 hour in the control group; or for <1 hour in the presence of feeding with cyclosporine (CyA). A direct in situ polymerase chain reaction (ISPCR) for the SRY gene showed SRY-marked endothelial and smooth muscle-like cells in neointima at 2 weeks in the PCI group, at 4 weeks in the control group, and rarely at 3 months in the CyA group. Staining by H&E showed the aortic graft intima to be thicker in the PCI than in the control group at 4 weeks, but thinning thereafter. The SRY-positive cells correlated with intimal thickness in the PCI and the control group ( r = .801 and .825; P < .05 and <.05, respectively), but not in the CyA group ( r = .247, P > .5). Our data suggest that prolonged cold preservation promotes recipient cell participation in graft arteriosclerosis after endothelium injury. The early neointimal formation via recipient cells incorporated into arteriosclerotic neointima may delay later intimal thickening. In the aortic allotransplantation model, prolonged cold ischemia may be beneficial for long-term graft survival due to early endothelial replacement. We hypothesize that controlled injury to the graft may serve as a new strategy for treatment of intimal thickening.