Daniel Larzul

Viral inactivation of human bone tissue using supercritical fluid extraction

A new bone tissue process using supercritical carbon dioxide fluid extraction (SFE) has been evaluated for its ability to inactivate or eliminate viruses. Four viruses, human immunodeficiency virus type 1 (HIV-1), Sindbis virus, polio Sabin type I virus, and pseudorabtes virus (PRV), were exposed to four different processing steps. In addition to supercritical CO2, hydrogen peroxide, sodium hydroxide, and ethanol treatments were evaluated. The mean cumulated reduction factors (log10) for the four viruses exposed to these four steps were > 14.2 for HIV-1, > 18.2 for Sindbis virus, > 24.4 for poliovirus, and > 17.6 for PRV. The mean reduction factors obtained by the supercritical fluid extraction alone were > 4.0, > 4.3, > 6.6, and > 4.0, respectively. These results demonstrate that the SFE process is effective in inactivating viruses on human femoral heads, and provides a level of inactivation similar to that obtained by traditional cleaning methods. It is proposed that CO2 SFE be incorporated as a routine step in the processing of bone allografts for transplantation either to replace or supplement existing procedures. ASAIO Journal 1998; 44:289–293.

Chronic non-B, non-C hepatitis among blood donors assessed with HCV third generation tests and polymerase chain reaction

Forty five blood donors with increased serum aminotransferases levels had liver histologic assessment and were tested for antibodies to hepatitis C virus (anti-HCV) with second and third generation ELISAs and RIBAs, and for HCV RNA with polymerase chain reaction (PCR) in serum and liver tissue. Twenty-nine of these 45 donors (65%) had steatosis without chronic hepatitis. Sixteen (35%) had chronic hepatitis. Twelve had evidence of HCV infection. Four had no evidence of HCV infection demonstrable by ELISA, RIBA or PCR. These four patients had no known cause of chronic hepatitis and no risk factor for parenterally acquired viral infection was found in them. This observation supports the hypothesis that a non-B, non-C virus might be implicated in chronic hepatitis. However, this hypothesis remains to be demonstrated.

A non-radioactive diagnostic test for the detection of HBV DNA sequences in serum at the single molecule level

A non-radioactive diagnostic test, using an acetylaminofluorene-labelled DNA probe, was developed to detect HBV DNA sequences in serum. In vitro enzymatic amplification was employed to increase the amount of HBV DNA sequences, and an amplification rate up to 1.5 x 10(7) was observed. When a dot-blot was performed after amplification with the Klenow fragment for 32 cycles, the detection limit was 3-30 particles. Sera from 20 blood donors and 10 HBs-Ag carriers were screened simultaneously, with the non-radioactive test performed after 28 amplification cycles, and with the classical radioactive test without amplification. An acceptable correlation was obtained between these two techniques. In Southern blot analysis of samples amplified with the thermoresistant DNA polymerase (Taq polymerase) for 40 cycles, a single DNA molecule was detected. Thermal treatment at 115 degrees C efficiently disrupted purified viral particles and allowed the detection of a single viral particle. Applied to crude serum, a kinetic study showed that this treatment was optimal after an incubation time of up to 10 min. Under these conditions, the detection limit was approximately 2 x 10(5) viral particles, after 40 amplification cycles performed with the Taq polymerase.

Non-radioactive hepatitis B virus DNA probe for detection of HBV-DNA in serum

A diagnostic test has been developed to detect hepatitis B virus (HBV) DNA in human sera. This test involves a dot-blot technique in which non-radioactive nucleic acid labelled with 2-acetylaminofluorene (AAF) is used as probe. Two series of human sera from 228 blood donors and 113 HBsAg chronic carriers were tested by hybridization with the same DNA probe labelled either with AAF or with 32P. A correlation between the techniques was observed for 328 sera (96%), and using the non-radioactive test it was possible to detect 56 (86%) of the 65 HBV-DNA-positive patients. A comparative study of the HBeAg/anti-HBe status and the presence of HBV-DNA was carried out on the sera from 113 HBsAg chronic carriers, 65 of which were positive for HBeAg and 29 of which were positive for anti-HBe antibodies. With the AAF test, 44 of the HBeAg-positive sera were positive, while 5 of the anti-HBe-positive sera were positive. This study shows that, although this non-radioactive test is slightly less sensitive than the radioactive hybridization assay (RHA), it can be used for a survey of HBV carriers. Dissociation of the viral multiplication and the HBe/anti-HBe status was identified with the AAF test as well as with the RHA. It would therefore appear that the AAF test described here may be used for the extensive survey of HBV multiplication.

Bone allografts and supercritical processing: effects on osteointegration and viral safety

Abstract A new bone tissue process using supercritical carbon dioxide extraction was evaluated for viral inactivation and the allografts produced by this process were tested in an in vivo implantation experiment. Four viruses, human immunodeficiency virus type I (HIV-1), Sindbis virus, Polio Sabin type I virus and Pseudorabies virus (PRV) were assayed. Four processing stages, supercritical CO2, hydrogen peroxide, sodium hydroxide and ethanol treatments were also tested. The efficiency of the process was assessed in terms of reduction factors which are the log10 of the ratio of the virus load before and after the stage to be evaluated. The cumulated reduction factors were the following: >18.2 for Sindbis virus, >24.4 for Poliovirus, >17.6 for PRV and >14.2 for HIV-1. Such allografts processed in this way were implanted into sheep leading to a much faster osseointegration in comparison with non-treated allografts. The combination of better graft incorporation and viral safety suggest that this process could become a new way for processing bank bones, alternatively or additionally, to the procedures presently used.

An automatic modified polymerase chain reaction procedure for hepatitis B virus DNA detection.

In order to perform an efficient and reproducible diagnostic test for hepatitis B virus (HBV) infection using the polymerase chain reaction (PCR), sixteen primer couples specific for the HBV genome were selected. Primers 15-31 nucleotides in length containing between 31-73% GC permitted amplification of fragments corresponding to the whole HBV genome. The specificity and efficiency of PCR amplification were studied in detail using DNA extracted from either a viral particle preparation or from the liver of a patient with chronic active hepatitis. Three primer couples in the X, C and PreS regions, i.e. MD24/MD26, MD27/MD31 and MD19/MD18, respectively, gave satisfactory results and performed efficiently under highly stringent hybridization conditions. A modified PCR procedure was then developed using only two thermal steps with a temperature shift of 16 degrees C. This simple method was as efficient as conventional PCR and permitted detection of a single HBV DNA molecule with the X region specific primer couple. The automatization of this PCR-based procedure permitted 40 amplification cycles in 105 min.

Polymerase chain reaction amplification of rabies virus nucleic acids from total mouse brain RNA

In an attempt to improve the sensitivity of the rabies genome hybridization test, PCR amplification was used following reverse transcription of rabies RNA extracted from infected brain. Presence of amplified DNA is demonstrated with either cDNA synthesized from the antigenomic primer or from antimessenger primer.