Viral validation design of a manufacturing process.

In many cases, the viral safety evaluation of biological products does not derive solely from direct testing for the presence of contaminants, but also from the demonstration that the manufacturing process is able to inactivate/eliminate them. This is achieved by the voluntary addition of a virus load at various steps of the process and the evaluation of viral reduction during the subsequent steps. The major difficulty for such viral validation studies is less to calculate a reduction factor for each step than to identify clearly and demonstrate the contribution of each in-process parameter to the reduction.

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 pseudorabies virus (PRV), were exposed to four different processing steps. In addition to supercritical CO2, hydrogen peroxide, sodium hydroxide, and ethanol treatments were evaluated.

Hepatitis C virus genotype 4 is highly prevalent in central Africa (Gabon).

Following a survey of hepatitis C virus (HCV) infection recently carried in central Africa (Gabon), we cloned and sequenced PCR products of the 5' non-coding and capsid-encoding regions of HCV RNA from three randomly selected HCV RNA-positive Gabonese subjects. In the capsid-encoding region, the identity between the three Gabonese isolates was 91 to 98%. The three Gabonese sequences showed a divergence of 11 to 17% from published HCV genotypes I to IV (1a, 1b, 2a and 2b) isolates and of 6 to 11% from HCV genotype 4 isolates.

Comparison of the sensitivity of nested PCR in the 5' non-coding and the NS5 regions of the HCV genome.

Thirty-seven patients with antibodies to hepatitis C virus detected by second-generation enzyme-linked immunoabsorbent assay were studied. Serum of 20 patients with increased serum alanine aminotransferase and 17 patients with normal serum alanine aminotransferase levels was tested for hepatitis C virus RNA with reverse transcription and nested polymerase chain reaction. The nested polymerase chain reaction was independently performed in both the 5' non-coding region and putative non-structural 5 region.

Prevalence of hepatitis C virus infection in asymptomatic anti-HIV1 negative pregnant women and their children.

The prevalence of hepatitis C virus (HCV) infection was studied prospectively in pregnant women in France and their children by detection of anti-HCV with second-generation ELISA (ELISA2). In ELISA2-positive women, anti-HCV was detected with second- and third-generation RIBA (RIBA2 and RIBA3) and serum HCV RNA was detected with PCR. Among 670 women, anti-HIV1-negative, 26 (3.

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.

Cryoglobulinemia with vasculitis associated with hepatitis C virus infection.

Essential mixed cryoglobulinemia is frequently associated with chronic hepatitis. This report presents four cases of cryoglobulinemia with vasculitis associated with chronic hepatitis related to hepatitis C virus infection. Hepatitis C virus infection was ascertained in the four patients by both the presence in the serum of anti-HCV antibodies detected by the four-antigen recombinant immunoblot assay and of HCV RNA detected by polymerase chain reaction.

The polymerase chain reaction: basic methodology and applications.

The "polymerase chain reaction" (PCR) is a high-power molecular biology technique allowing in vitro enzymatic amplification of a given DNA sequence. This exponential amplification can reach 10(7)-10(9), even a single DNA molecule can be detected. Also the use of non-radioactive probes, considered to be less sensitive than their radioactive counterparts, is possible for the molecular hybridization, to retain a high level of sensitivity.

Isolation of HIV-1 from seropositive people living in Cotonou, Benin.

Benin is located in West Africa and is situated between HIV-2 and HIV-1-endemic zones. The first cases of HIV-1 infection in Benin were reported in 1987. Since then, AIDS cases have been diagnosed there and the number of known HIV-seropositive people has rapidly increased.

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.

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.

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.

Identification and classification of Campylobacter strains by using nonradioactive DNA probes.

Acetylaminofluorene-labeled genomic DNA probes were used for the identification and classification of Campylobacter strains. Relationships among 17 well-known strains of Campylobacter species and subspecies were studied by comparing acetylaminofluorene- or 32P-labeled probes. Results obtained with both methods were closely correlated and were in agreement with those already reported.

Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification.

In vitro enzymatic amplification was applied to detect hepatitis B virus (HBV) DNA sequences in serum. This technique, known as the polymerase chain reaction (PCR) was used to amplify a 128 bp DNA fragment including a 112 nucleotide long sequence complementary to a region in the S gene of the HBV genome. Amplified samples were subjected to spot-test hybridization and scintillation counting using a 32P-labeled oligonucleotide probe.

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.