European collaborative evaluation of the Enzygnost HBsAg 6.0 assay: performance on hepatitis B virus surface antigen variants.

Amino acid changes within the major antigenic determinant of the hepatitis B virus (HBV) surface antigen (HBsAg) may modify eventually the antigenic properties of the protein and may have impact on the sensitivity of diagnostic assays. Modifications in the design of an assay can, however, improve significantly its ability to detect HBV mutants. One hundred forty-seven clinical samples containing HBsAg variants, and 54 supernatants of cells expressing recombinant HBsAg mutants were tested by two generations of a commercial HBsAg test (Enzygnost® HBsAg 5.0 and 6.0, Siemens Healthcare Diagnostics Products, Marburg, Germany), and the results were compared. A significant improvement was demonstrated for the second test by comparing the mean and individual sample/cut-off values, as well as by the detection of several samples displaying amino acid changes in residues 120 and 145 of the HBsAg which were recorded as negative by the former test. The results showed that modifications in design of the assay improved considerably the ability of the test to detect HBsAg mutants, and that difficulties in detecting such HBV variants should not be expected with the routine use of the test in diagnostic laboratories and in blood transfusion centers.

Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30.

Here we report recovery of infectious Marburg virus (MARV) from a full-length cDNA clone. Compared to the wild-type virus, recombinant MARV showed no difference in terms of morphology of virus particles, intracellular distribution in infected cells, and growth kinetics. The nucleocapsid protein VP30 of MARV and Ebola virus (EBOV) contains a Zn-binding motif which is important for the function of VP30 as a transcriptional activator in EBOV, whereas its role for MARV is unclear. It has been reported previously that MARV VP30 is able to support transcription in an EBOV-specific minigenome system. When the Zn-binding motif was destroyed, MARV VP30 was shown to be inactive in the EBOV system. While it was not possible to rescue recombinant MARV when the VP30 plasmid was omitted from transfection, MARV VP30 with a destroyed Zn-binding motif and EBOV VP30 were able to mediate virus recovery. In contrast, rescue of recombinant EBOV was not supported by EBOV VP30 containing a mutated Zn-binding domain.

The Ebola virus genomic replication promoter is bipartite and follows the rule of six.

In this work we investigated the cis-acting signals involved in replication of Ebola virus (EBOV) genomic RNA. A set of mingenomes with mutant 3' ends were generated and used in a reconstituted replication and transcription system. Our results suggest that the EBOV genomic replication promoter is bipartite, consisting of a first element located within the leader region of the genome and a second, downstream element separated by a spacer region. While proper spacing of the two promoter elements is a prerequisite for replication, the nucleotide sequence of the spacer is not important. Replication activity was only observed when six or a multiple of six nucleotides were deleted or inserted, while all other changes in length abolished replication completely. These data indicate that the EBOV replication promoter obeys the rule of six, although the genome length is not divisible by six. The second promoter element is located in the 3' nontranslated region of the first gene and consists of eight UN5 hexamer repeats, where N is any nucleotide. However, three consecutive hexamers, which could be located anywhere within the promoter element, were sufficient to support replication as long as the hexameric phase was preserved. By using chemical modification assays, we could demonstrate that nucleotides 5 to 44 of the EBOV leader are involved in the formation of a stable secondary structure. Formation of the RNA stem-loop occurred independently of the presence of the trailer, indicating that a panhandle structure is not formed between the 3' and 5' ends.

Ebola virus VP30-mediated transcription is regulated by RNA secondary structure formation.

The nucleocapsid protein VP30 of Ebola virus (EBOV), a member of the Filovirus family, is known to act as a transcription activator. By using a reconstituted minigenome system, the role of VP30 during transcription was investigated. We could show that VP30-mediated transcription activation is dependent on formation of a stem-loop structure at the first gene start site. Destruction of this secondary structure led to VP30-independent transcription. Analysis of the transcription products of bicistronic minigenomes with and without the ability to form the secondary structure at the first transcription start signal revealed that transcription initiation at the first gene start site is a prerequisite for transcription of the second gene, independent of the presence of VP30. When the transcription start signal of the second gene was exchanged with the transcription start signal of the first gene, transcription of the second gene also was regulated by VP30, indicating that the stem-loop structure of the first transcription start site acts autonomously and independently of its localization on the RNA genome. Our results suggest that VP30 regulates a very early step of EBOV transcription, most likely by inhibiting pausing of the transcription complex at the RNA structure of the first transcription start site.