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Fourth-generation assays for the simultaneous detection of human immunodeficiency virus (HIV) antigenSince the first enzyme immunoassays (EIA) for blood donor screening and laboratory diagnosis of human immunodeficiency virus (HIV) infection were licensed over 15 years ago, the quality of these tests has been continuously improved by the use of recombinant antigens and synthetic peptides (second test generation) and the sandwich EIA technology (third test generation) (10, 36). There is however a residual risk for false-negative results. The potential causes include the diagnostic window in the preseroconversion phase, genetic variability, atypical seroconversions, a delayed or absent immune re-

In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.

It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in protein p54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the p54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against p54 protein. The sequence of this recombinant phage allowed the location of the p54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a protein of 183 amino acids with a calculated molecular weight of 19,861. This protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for protein processing of several ASFV structural proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the p54 gene is translated from a late mRNA and the protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the p54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when p54 genes from viral clones generated by cell culture passage and coding for p54 proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in p54, resulting in stepwise modifications in the molecular weight of the protein. These duplications and deletions of a tandem repeat sequence array within a protein coding region constitute a novel mechanism of genetic diversification in ASFV.