Objective The purpose of our work was to examine the most reliable laboratory diagnosis of fetal parvovirus B19 infection in hydropic fetuses by evaluating the most appropriate clinical sample and laboratory test. Design B19 DNA detection in fetal samples and serological signs of B19 infection in the respective mothers. Samples collected between January 2000 and July 2008. Setting Microbiology, University of Bologna, Bologna, Italy. Samples One hundred thirty‐five fetal samples (58 fetal cord blood and 77 amniotic fluid samples) and 109 serum samples collected from 109 pregnant women. Methods Validated and certified in situ hybridisation assay (ISH) and polymerase chain reaction–enzyme‐linked immunosorbent assay (PCR‐ELISA) were performed on fetal samples to detect B19 DNA. B19‐specific antibodies were investigated in maternal serum samples by a commercial enzyme immunoassay. Main outcome measures Parvovirus B19 DNA detection in fetal specimens was analysed in relation to maternal serological signs of infection. Results Parvovirus B19 DNA was detected in 22.41% of fetal cord blood and 36.36% of amniotic fluid samples. A statistically significant difference was found between DNA detection by ISH (23.70%) and PCR‐ELISA (14.81%) ( P = 0.004). Only 11.76% of fetuses with virological diagnosis of B19 infection were from women with serological signs of acute/recent B19 infection. Conclusions Diagnosis of fetal parvovirus B19 infection cannot always rely on maternal serological investigations but rather on the virological analysis of fetal samples. Both fetal cord blood and amniotic fluid samples are suitable for diagnosis, but the detection of B19 DNA in the cells of amniotic fluid samples by ISH proved to be the most reliable diagnostic system.
In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.
We describe a simple and rapid dot immunoperoxidase assay for the direct detection of parvovirus B19 capsid antigens in human sera. The assay was performed with serum specimens dotted onto nylon membranes. VP1 and VP2 B19 antigens, which represent 4 and 96% of the capsid, respectively, were detected with a pool of monoclonal antibodies directed against the two proteins, and the complex was visualized by immunoperoxidase staining. The assay could be performed in about 4 h, and positive results were revealed at the end of the reaction as dark blue spots on the nylon membrane at the site of positive specimens. A total of 541 serum samples from different subjects and with different laboratory evaluations with regard to B19 infection were analyzed. The results obtained by the dot immunoperoxidase assay were compared with the results obtained for the presence of B19 DNA by dot blot hybridization and nested PCR. With optimized working conditions, the dot immunoperoxidase assay was able to detect the presence of B19 with a sensitivity comparable or slightly higher than that achieved by dot blot hybridization but less than that achieved by nested PCR. Since the level of sensitivity of the dot immunoperoxidase assay proved to be appropriate for the detection of acute B19 infection, and since the cost, time to a result, and versatility of the assay are important issues, from our evaluation, the dot immunoperoxidase assay described may be particularly suitable for large-scale screening of samples and a good alternative to DNA detection methods in the routine laboratory evaluation of B19 infection.
Abstract In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.
Human Cytomegalovirus (HCMV) was inoculated in synchronized human fibroblasts at different phases of the cell cycle. the virus replication appeared strongly dependent on the host metabolic state, being faster when the infection was carried out in G2 and S phase.
SUMMARY The effect of heat shock was investigated on lymphoblastoid cell lines Raji and P3HR1 harbouring Epstein-Barr virus (EBV) genomes, and on Vero cells abortively infected with human cytomegalovirus (HCMV). A heat shock at 44 °C for 10 min induced the appearance of EBV early antigens in Raji cells and increased the percentage of cells expressing EBV viral capsid antigens in P3HR1 cells. Heat shock performed on Vero cells just before HCMV infection resulted in an approximately fourfold increase in the number of cells exhibiting early nuclear antigens, and in the appearance of HCMV-induced Fc receptors.
Leung, D. YM.; Meissner, H. C.; Fulton, D. R.; Murray, D. L.; Kotzin, B. L.; Schlievert, P. M.; Nigro, G.; Zerbini, M..; Yoto, Y.; Kudoh, T.; Cohen, B. J.
SUMMARY The study of human cytomegalovirus (HCMV) in cultures of human embryo lung fibroblasts, pre-treated with actinomycin D, has shown that under these conditions the virus infection does not proceed beyond the ‘early’ events of the virus replication cycle. In the same experimental conditions the growth of poliovirus type 1, vaccinia virus and herpes simplex type 1 virus, was completely unaffected. These results suggest that the complete HCMV replication cycle requires some cellular function(s) between early transcription of the input virus genome and virus DNA synthesis.
