Development and use of an internal standard for oyster herpesvirus 1 detection by PCR.

Oyster samples were examined using a competitive PCR method in order to detect and quantify oyster herpesvirus 1 (OsHV-1) DNA. Quantitation of viral DNA by competitive PCR assay was based on co-amplification of OsHV-1 DNA and a competitor where a known amount of competitor DNA was present in the same reaction mixture. The competitor was engineered so that it differs in length (deletion of 76 base pairs) from the viral DNA. The assay allowed the detection of 1 fg of viral DNA among 0.5 mg of oyster tissues. The method was used to demonstrate the absence of PCR inhibitors in oyster spat ground tissues. PCR inhibition was observed in adult oyster samples when the same tissue preparation procedure was used. On the contrary, classical phenol/chloroform DNA extraction from adult oyster tissues allowed co-amplification of the internal standard competitor and the viral DNA. The method was successfully used to demonstrate the presence of viral DNA in asymptomatic adult oysters. Quantitation of OsHV-1 DNA in infected spat and asymptomatic adult oysters was also carried out. Viral DNA (1.5-325 pg) were detectable in 0.5 mg of oyster tissues in adults. The amounts of viral DNA in infected oyster spat varied from 750 pg to 35 ng per 0.5 mg of ground tissues.

Herpes-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes.

An in situ hybridization protocol for detecting the herpes-like virus which infects French Pacific oysters, Crassostrea gigas, was developed. Two DNA probes were synthesized by incorporation of digoxigenin 11-dUTP during PCR. Two oyster herpes-like virus specific primer pairs, A5/A6 and C1/C6, were used. Both DIG-labelled probes were able to detect 50 pg of herpes-like virus PCR amplified DNA in Southern blot hybridizations. The probes hybridized with viral DNA in paraffin sections of infected C. gigas spat. No non-specific binding was observed. The ability of the defined in situ hybridization technique to diagnose herpes-like virus infections in oysters was compared with light and transmission electron microscopy techniques in infected and non-infected spat. In situ hybridization assays were also conducted on paraffin sections to determine virus distribution within the host and to study the pathogenesis infection. In situ hybridization confirmed that the expression pattern of the herpes-like virus was restricted to connective tissues as described previously by light and transmission electron microscopy. However, this technique also allowed the detection of viral DNA in the oyster nervous system. Some labelled cells were observed in the visceral ganglion of infected oyster spat.

Experimental herpes-like viral infections in marine bivalves: demonstration of interspecies transmission.

Since 1972, herpes-like virus infections have been reported in several marine bivalve species around the world. Viral detection was often associated with high mortality rates in larvae and spat. To determine whether a single virus is able to infect different bivalve host species, we carried out experimental transmission assays. As a first step, 8 assays were performed to infect axenic Crassostrea gigas larvae with virus from infected C. gigas larvae using a previously described protocol. The protocol appeared reliable and PCR was confirmed as a powerful technique for detecting viral DNA in experimentally infected oysters. The defined protocol was then applied to infect different bivalve species. Interspecies viral transmission was demonstrated under laboratory conditions. The same phenomenon may occur in private hatcheries and may be promoted by intensive rearing conditions. This hypothesis is reinforced by reports of concomitant mortalities in the larvae of several bivalve species and by the first molecular analysis of infected larval samples.

A herpes-like virus infects a non-ostreid bivalve species: virus replication in Ruditapes philippinarum larvae.

Sporadic high mortalities were reported in June 1997 among hatchery-reared larval Manila clam Ruditapes philippinarum in a French commercial hatchery. Cellular abnormalities were observed in semi-thin sections in affected animals. Transmission electron microscopy revealed the presence of herpes-like virus particles in larvae. This is the first description of a herpes-like virus infection in larval R. philippinarum, a non-ostreid bivalve species. Virus particles were similar to other herpes-like viruses described from different oyster species with respect to ultrastructure and morphogenesis. Electron microscopic examination also demonstrated cells with condensed chromatin and extensive perinuclear fragmentation of chromatin. Like viruses infecting oysters, the herpes-like virus detected in clams may induce apoptosis in infected animals.

Development of a PCR procedure for the detection of a herpes-like virus infecting oysters in France.

A PCR-based procedure for detecting a herpes-like virus that infects the Japanese oyster, Crassostrea gigas, in France was developed. Two primers were designed to provide specific amplification products ranging in size from 917 to 1001 bp when carried out on oyster herpes-like virus DNA. No amplification was observed of oyster genomic DNA nor of the DNA from vertebrate herpesviruses. Crude samples were prepared and submitted to nested PCR, allowing amplification of DNA fragments of the expected size when carried out on infected larval and spat samples. The procedure used to prepare the sample for PCR was found to be critical because of the presence of unidentified substances in oyster tissues that inhibit the PCR reaction. A rapid and convenient sample preparation using ground tissues allowed a sensitive detection of the herpes-like virus infected oysters. The ability of the defined PCR protocol to diagnose herpes-like virus infections in oysters was compared to the transmission electron microscopy technique using 15 C. gigas larval batches with or without mortalities. PCR amplification is as sensitive a diagnostic assay for herpes-like virus as transmission electron microscopy. However, the nested PCR protocol is more convenient and less time consuming. The relationship between reported mortalities among C. gigas oyster spat and herpes-like virus DNA detection by PCR was also investigated. Statistical analysis showed that virus detection and mortalities are correlated. This observation highlights the importance of studying the causative role of herpes-like virus in oyster spat mortalities.

Detection of disseminated tumor cells in peripheral blood of colorectal cancer patients.

All cancer staging systems seek to identify clinical and pathological features that can predict outcome or guide therapy. In particular, a non-invasive method for the early detection of disseminating disease would be of great interest. We investigated the use of cytokeratin genes expression to detect blood metastases from colorectal tumors. Epithelial tumor cells were isolated from whole blood using the monoclonal antibody (MAb) BerEP4 and magnetic beads, and detected by reverse transcription-polymerase chain reaction using oligonucleotides derived from the cDNA sequences of cytokeratins 8, 19 and 20. The sensitivity of this assay was determined by spiking SW620 colon carcinoma cells in normal blood. Using cytokeratin 19 expression we were able to detect 1 epithelial tumor cell in 1 ml of whole blood. The clinical applicability of this technique was explored by evaluating patients with a colorectal carcinoma. Epithelial cells were detected in the blood of 12 out of 23 patients, 2 (20%) of 10 with Astler-Coller stage A or B, and 10 (77%) of 13 with stage C or D cancer. In conclusion, this test is a non-invasive, sensitive, and specific assay for detecting circulating epithelial cells in blood. It may be useful for the early diagnosis of disseminating disease, to determine whether the presence of micrometastatic cells at the time of surgery is correlated with an early relapse and for monitoring adjuvant therapeutic trials.