Varicella-zoster virus DNA in cells isolated from human trigeminal ganglia

To determine the type of cell(s) that contain latent varicella-zoster virus (VZV) DNA, we prepared pure populations of neurons and satellite cells from trigeminal ganglia of 18 humans who had previously had a VZV infection. VZV DNA was present in 34 of 2,226 neurons (1.5%) and in none of 20,700 satellite cells. There was an average of 4.7 (range of 2 to 9) copies of VZV DNA per latently infected neuron. Latent VZV DNA was primarily present in large neurons, whereas the size distribution of herpes simplex virus DNA was markedly different.     

Molecular survey of latent pseudorabies virus infection in nervous tissues of slaughtered pigs by nested and real-time PCR

In this study, the prevalence and quantity of a latent pseudorabies virus (PrV) infection in the nervous tissues of randomly selected pigs was determined via nested and real-time PCR. The nervous tissues, including the trigeminal ganglion (TG), olfactory bulb (OB), and brain stem (BS), were collected from the heads of 40 randomly selected pigs. The majority of the nervous tissues from the selected pigs evidenced a positively amplified band on nested PCR. In particular, nested PCR targeted to the PrV glycoprotein B (gB) gene yielded positive results in all of the BS samples. Nested PCR for either the gE or gG gene produced positive bands in a less number of nervous tissues (57.5% and 42.5%, respectively). Real-time PCR revealed that the examined tissues harbored large copy numbers of latent PrV DNA, ranging between 10(0.1) and 10(7.2) (1-1.58 x 10(7)) copies per 1 microg of genomic DNA. Real-time PCR targeted to the PrV gE gene exhibited an accumulated fluorescence of reporter dye at levels above threshold, thereby indicating a higher prevalence than was observed on the nested PCR (100% for BS, 92% for OB, and 85% for TG). These results indicate that a large number of farm-grown pigs are latently infected with a field PrV strain with a variety of copy numbers. This result is similar to what was found in association with the human herpes virus.     

Quantitation of latent varicella-zoster virus DNA in human trigeminal ganglia by polymerase chain reaction.

Competitive polymerase chain reaction was used to quantitate latent varicella-zoster virus (VZV) DNA in human trigeminal ganglia. Ganglionic DNA from five subjects was amplified with oligonucleotide primers specific for VZV gene 28. Two of the samples were also analyzed with primers specific for VZV gene 62. Our results indicated that there are 6 to 31 copies of the VZV genome in every 100,000 ganglionic cells.     

The high prevalence of herpes simplex virus type 1 DNA in human trigeminal ganglia is not a function of age or gender

The purpose of this study was to determine the presence and copy numbers of herpes simplex virus type 1 (HSV-1) DNA in human trigeminal ganglia (TG) with respect to age, gender, and postmortem interval (PMI). Human TG (n = 174, obtained from the Oregon Brain Bank, with data on age, gender, and PMI) were analyzed for HSV-1 DNA copies (HSV-1 DNA polymerase gene) by using real-time PCR. We found that 89.1% (131/147) of subjects and 90.1% (155/174) of TG contained HSV-1 DNA. The copy numbers of HSV-1 DNA in the positives ranged from very high (>10⁶) to very low (5). These data confirm and strengthen our previous findings that subjects were positive for HSV-1 DNA in tears (46/50; 92%) and saliva (47/50; 94%). These TG data and tear and saliva data demonstrated considerable variability in copy numbers of HSV-1 DNA per subject. Statistical analysis showed no significant relationship between gender and copy number, age and copy number, or PMI and copy number for each pair of variables. A factorial analysis of gender, age, and PMI with respect to copy number also showed no statistical significance. This is the first study that provides statistical analysis that documents that the prevalence of HSV-1 DNA in the human TG is not a function of either gender or age.     

Replication of herpes simplex virus type 1 within trigeminal ganglia is required for high frequency but not high viral genome copy number latency

The replication properties of a thymidine kinase-negative (TK(-)) mutant of herpes simplex virus type 1 (HSV-1) were exploited to examine the relative contributions of replication at the body surface and within trigeminal ganglia (TG) on the establishment of latent infections. The replication of a TK(-) mutant, 17/tBTK(-), was reduced by approximately 12-fold on the mouse cornea compared to the rescued isolate 17/tBRTK(+), and no replication of 17/tBTK(-) in the TG of these mice was detected. About 1.8% of the TG neurons of mice infected with 17/tBTK(-) harbored the latent viral genome compared to 23% of those infected with 17/tBRTK(+). In addition, the latent sites established by the TK(-) mutant contained fewer copies of the HSV-1 genome (average, 2.3/neuron versus 28/neuron). On the snout, sustained robust replication of 17tBTK(-) in the absence of significant replication within the TG resulted in a modest increase in the number of latent sites. Importantly, these latently infected neurons displayed a wild-type latent-genome copy number profile, with some neurons containing hundreds of copies of the TK(-) mutant genome. As expected, the replication of the TK(-) mutant appeared to be blocked prior to DNA replication in most ganglionic neurons in that (i) virus replication was severely restricted in ganglia, (ii) the number of neurons expressing HSV proteins was reduced 30-fold compared to the rescued isolate, (iii) cell-to-cell spread of virus was not detected within ganglia, and (iv) the proportion of infected neurons expressing late proteins was reduced by 89% compared to the rescued strain. These results demonstrate that the viral TK gene is required for the efficient establishment of latency. This requirement appears to be primarily for efficient replication within the ganglion, which leads to a sixfold increase in the number of latent sites established. Further, latent sites with high genome copy number can be established in the absence of significant virus genome replication in neurons. This suggests that neurons can be infected by many HSV virions and still enter the latent state.     

Quantitative detection of the free-living amoeba Hartmannella vermiformis in surface water by using real-time PCR

A real-time PCR-based method targeting the 18S rRNA gene was developed for the quantitative detection of Hartmannella vermiformis, a free-living amoeba which is a potential host for Legionella pneumophila in warm water systems and cooling towers. The detection specificity was validated using genomic DNA of the closely related amoeba Hartmannella abertawensis as a negative control and sequence analysis of amplified products from environmental samples. Real-time PCR detection of serially diluted DNA extracted from H. vermiformis was linear for microscopic cell counts between 1.14 x 10(-1) and 1.14 x 10(4) cells per PCR. The genome of H. vermiformis harbors multiple copies of the 18S rRNA gene, and an average number (with standard error) of 1,330 +/- 127 copies per cell was derived from real-time PCR calibration curves for cell suspensions and plasmid DNA. No significant differences were observed between the 18S rRNA gene copy numbers for trophozoites and cysts of strain ATCC 50237 or between the copy numbers for this strain and strain KWR-1. The developed method was applied to water samples (200 ml) collected from a variety of lakes and rivers serving as sources for drinking water production in The Netherlands. Detectable populations were found in 21 of the 28 samples, with concentrations ranging from 5 to 75 cells/liter. A high degree of similarity (> or =98%) was observed between sequences of clones originating from the different surface waters and between these clones and the reference strains. Hence, H. vermiformis, which is highly similar to strains serving as hosts for L. pneumophila, is a common component of the microbial community in fresh surface water.     

Productive varicella-zoster virus infection of cultured intact human ganglia

Varicella-zoster virus (VZV) is a species-specific herpesvirus which infects sensory ganglia. We have developed a model of infection of human intact explant dorsal root ganglia (DRG). Following exposure of DRG to VZV, viral antigens were detected in neurons and nonneuronal cells. Enveloped virions were visualized by transmission electron microscopy in neurons and nonneuronal cells and within the extracellular space. Moreover, rather than remaining highly cell associated during infection of cultured cells, such as fibroblasts, cell-free VZV was released from infected DRG. This model enables VZV infection of ganglionic cells to be studied in the context of intact DRG.     

NEURONAL AND NEUROPIL FRACTIONS FROM DEVELOPING RAT BRAIN

Abstract— A method is described for the preparation of enriched fractions containing isolated neuronal and glial cells from brains derived from 1 to 20-day-old rats. The method is based on mechanical disaggregation in a medium containing Ficoll-PVP followed by centrifugation on a single-stage two-step gradient at 13,000 g for 30min. The neuronal and neuropil (glial) fractions are approx 70–80% pure in cellular terms.
The cells showed well-preserved cytoplasmic and nuclear morphology at the light and electron microscope level and between 70 and 80% excluded trypan blue. Despite changes in the total cell population with age due to glial proliferation, the proportionate recovery of cells in the separated fractions was fairly constant: based on DNA determination, 23 and 29% of all neurons and 15 and 17% of glia were recovered in the purified fractions from Day 1 and Day 20 animals respectively.
Changes in neuronal cell size with age were reflected in a 2.5-fold increase in protein recovered in the neuronal fraction per mg DNA. Protein and RNA levels/mg DNA in the neuropil fraction reached a maximum at Day 10. It is concluded that the method produces a defined and reliable purification of cells in the separated fractions throughout the studied age range and therefore provides a sound basis for studies on the distribution of biochemical systems between cell types during post-natal development.     

Quantitation of latent varicella-zoster virus and herpes simplex virus genomes in human trigeminal ganglia

Using real-time fluorescence PCR, we quantitated the numbers of copies of latent varicella-zoster virus (VZV) and herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) genomes in 15 human trigeminal ganglia. Eight (53%) and 1 (7%) of 15 ganglia were PCR positive for HSV-1 or -2 glycoprotein G genes, with means of 2,902 +/- 1,082 (standard error of the mean) or 109 genomes/10(5) cells, respectively. Eleven of 14 (79%) to 13 of 15 (87%) of the ganglia were PCR positive for VZV gene 29, 31, or 62. Pooling of the results for the three VZV genes yielded a mean of 258 +/- 38 genomes/10(5) ganglion cells. These levels of latent viral genome loads have implications for virus distribution in and reactivation from human sensory ganglia.     

Recognition of individual genes in a single bacterial cell by fluorescence in situ hybridization--RING-FISH

Fluorescence in situ hybridization (FISH) using rRNA targeted oligonucleotide probes is a standard method for identification of microorganisms in environmental samples. Apart from its value as a phylogenetic marker ribosomal RNA has always been the favoured target molecule for FISH because of its abundance in all cells, whereas plasmids and DNA were regarded as unsuitable targets because of their low copy number. Here we present an improved FISH technique, which is based on polynucleotide probes. It goes beyond the detection of high copy intracellular nucleic acids such as rRNA (up to 10(4)-10(5) copies per cell) and allows for the first time the in situ detection of individual genes or gene fragments on plasmids (10(1)-10(3) copies per cell) and chromosomal DNA (<10 copies per cell) in a single cell. Using E. coli as model organism we were able to detect in situ cells harbouring the antibiotic resistance gene beta lactamase on high, medium and low copy plasmids as well as the chromosomal encoded housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, we detected the prepilin peptidase gene xpsO in the plant pathogen Xanthomonas campestris in situ. Because of the characteristic hybridization signal obtained with this method--a halo-like, ring-shaped concentration of fluorescence in the cell periphery--we coined the term RING-FISH (recognition of individual genes) to differentiate it from conventional FISH.     

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.