[Emerging viral diseases in Europe]. / "Neue" Viren als bedrohliche Krankheitserreger in Europa.

Emergence of viral agents in Europe is influenced by various factors. Climatic changes influencing possible vectors, insufficient vaccination, and travel of man and goods are among the most important reasons to explain these changes. Fever and arthralgia are the leading symptoms in infection with Dengue, Sindbis, or Chikungunya virus. In contrast, tick-born encephalitis (TBE), Toscana, or West Nile virus infections mainly lead to meningo-encephalitis. In Europe, hemorrhagic fever is caused by Crimean Congo and Hanta virus. Protective vaccines are available for emerging viral agents like TBE, influenza and measles.

Human immunodeficiency virus type-1 group M quasispecies evolution: diversity and divergence in patients co-infected with active tuberculosis.

The evolution of intra-host human immunodeficiency virus type 1 (HIV-1) quasispecies prior and after treating active tuberculosis (TB) with chemotherapy in HIV-1/TB patients was assessed. Two time points HIV-1 quasispecies were evaluated by comparing HIV-1-infected patients with active tuberculosis (HIV-1/TB) and HIV-1-infected patients without tuberculosis (HIV-1/non-TB). Plasma samples were obtained from the Frankfurt HIV cohort, and HIV-1 RNA was isolated. C2V5 env was amplified by PCR and molecular cloning was performed. Eight to twenty-five clones were sequenced from each patient. Various phylogenetic analyses were performed. We found a significant increase in diversity and divergence in HIV-1/TB compared to the HIV-1/non-TB. For HIV-1/TB, the average rate of evolution of C2V5 env was higher than previous reports (2.4 × 10(-4) substitution/site/day). Two groups of HIV-1/TB were observed based on the rate of HIV-1 evolution and coreceptor usage: A fast evolving R5-tropic dominating group and a relatively slowly evolving X4 group. The results demonstrated that active TB has an impact on HIV-1 viral diversity and divergence over time. The influence of active TB on longitudinal evolution of HIV-1 may be predominant for R5 viruses.

Various types of HIV mixed infections in Cameroon.

In order to assess the incidence of HIV mixed infection as well as to clarify the molecular epidemiology of HIV in central Africa, we investigated 43 HIVs obtained from 211 Cameroonian AC, ARC, and AIDS patients in 1994 and 1995. Part of the pol region and part of the env region were phylogenetically analyzed. The genotypes observed were varied: of 43 specimens, 28 (65%) were subtype A, 1 (2%) was subtype B, 2 (5%) were subtype D, 3 (7%) were subtype F, and 2 (5%) were group O. Of the remaining 7 specimens, 3 were mixed infections with HIV-1 subtypes A and C, HIV-1 subtypes C and F, and HIV-2 subtype A and HIV-1 subtype A; 1 was a mixed infection with HIV-1 subtypes A and D and the highly divergent group O (triple infection); another 3 appeared to consist of mosaic genomes (A/G, A/E, and B/A recombinant). These data show that various types of mixed infection, such as between different subtypes of HIV-1 group M, between HIV-1 and HIV-2, and even between HIV-1 groups O and M, were confirmed at a rather high frequency (approximately 10%). The mixed infection is particularly significant where there is a greater variety of HIV-1 subtypes circulating, since it results in new genetic diversity generated by intersubtype recombination.

Strongly enhanced sensitivity of a direct anti-HIV-1/-2 assay in seroconversion by incorporation of HIV p24 ag detection: a new generation vironostika HIV Uni-Form II.

The clinical sensitivity of the current anti-HIV assays is based for an important part on their reactivity with seroconversion panels. The most sensitive assay closes the seroconversion window as much as possible, thereby reducing the risk of transmitting false negative donations obtained from individuals infected recently. Because of the absence of anti-HIV antibodies during the early phase of infection, the seroconversion window can be narrowed partially by detection of HIV p24 Ag. To achieve this, the highest affinity anti-p24 binding antibodies were selected with BlAcore and applied in a direct assay format. To achieve optimal conditions for the anti-HIV part of the assay the HIV specific antigens viral HIV-1 gp160, HIV-2 gp36 and HIV-1 group O gp41 peptides were used. These antigens and antibodies were applied for microELISA coating as well as in the conjugate pearl, which is present in the well of the microELISA plate. The (analytical) anti-HIV-1/-2 and anti-HIV-1 group O sensitivity of this new assay, Vironostika HIV Uni-Form II Ag/Ab, is at least at the level of the current Vironostika HIV Uni-Form II plus O. When compared to the Vironostika HIV Uni-Form II plus O, the seroconversion window is narrowed by 1-2 weeks due to the incorporation of HIV p24 Ag detection. The level of reactivity of the anti-HIV and HIV Ag detection part can be improved by about a factor 2 by applying continuous shaking during sample incubation. Initial studies suggested that the specificity of the assay is identical to that of the Vironostika HIV Uni-Form II plus O, namely > 99.9%. Monitoring of proper execution of the assay handling steps was facilitated by implementing a purple dye in the conjugate pearl. Colourless specimen diluent changes into a green fluid upon dissolving of the conjugate pearl and turns subsequently into blue/purple upon sample addition. These visual changes can also be determined by spectrophotometric measurement at 620 nm.

Envelope sequence variability and serologic characterization of HIV type 1 group O isolates from equatorial guinea.

Four sera from Equatorial Guinea (EG) suspected to contain antibody against HIV-1 group O-related viruses were identified on the basis of unusual and differential serologic reactivity in selected commercial assays and Western blot. Degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences from the suspect EG sera. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. Analysis (PHYLIP package of programs) of Env amino acid sequences (translated from nucleotide sequences) indicated that the amino acid sequences obtained from EG sera clustered more closely with HIV Env sequences of group O compared to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY (one isolate), RIGPMAWY (two isolates), or GLGPLAVY (one isolate). The V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV (Los Alamos) database, but was present in two of our group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from three of the sera, RLLALETLIQNQQLLNLWGCKGR(K)L(I)VCYTSVK(T)W, whereas sequence from the fourth serum contained three changes as noted in parentheses. IDR sequences derived from EG sera were unique compared to those reported for other HIV-1 group O isolate ANT70, VAU, or MVP5180. Antibody in each EG serum directed against the IDR could be detected using synthetic peptides comprising sequences from the ANT70 or MVP5180 IDRs, but were most reactive against the sequences derived from the samples themselves. Little or no serologic reactivity was detected when EG sera were reacted against peptides comprising the IDR of HIV-1 group M (subtype B consensus) or HIV-2 (consensus).

PIP: The genetic variation and epidemiology of HIV-1 group O isolates are of considerable importance to the design of HIV-1 diagnostic and screening assays, especially since current serologic and genetic methods to detect HIV-1 have been developed mainly on the basis of sequences from isolates belonging to HIV-1 group M. The HIV envelope protein, especially the gp41 immunodominant region, plays a major antigenic role in the detection of HIV infection and for discriminating HIV-1 from HIV-2 antibody. This paper reports upon genetic variation and the serologic characterization of env sequences from 4 people living in Equatorial Guinea (EG) who were infected with HIV-1 group O. Selected commercial assays and Western blot were first used to identify the sera, then degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. The env amino acid sequence analysis found the EG sera sequences to be clustered more closely with the HIV env sequences of group O rather than to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY, RIGPMAWY, or GLGPLAVY. Although the V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV database, it was present in 2 of the group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from 3 of the sera. IDR sequences derived from the EG sera were unique compared to those reported for other HIV-1 group O isolates ANT70, VAU, or MVP5180. Other findings are discussed in detail.

Diversity of the immunodominant epitope of gp41 of HIV-1 subtype O and its validity for antibody detection.

The immunodominant regions of the gp41 from 13 HIV-1 subtype O strains from Cameroon, 11 from France and one from Germany were sequenced. The amino acid sequences were compared to those of the 3 published HIV-1 subtype O isolates, ANT70, MVP-5180 and VAU. All HIV-1 subtype O isolates had a very conserved amino acid sequence in this region and showed a subtype O specific structure. Within the cysteine loop there was a positive charge of two basic amino acids, arginine and lysine. Only two strains (CM.6778 and CM.8161) showed an acidic amino acid in this loop. None of the isolates showed the same amino acid sequence in this immunodominant region. A 25 residue peptide from the immunodominant domain of gp41 of the MVP-5180 strain was synthesized, cycled to form the cysteine-loop and coated to microtiter plates. Antibody binding was detected by indirect ELISA using an enzyme labeled anti-human IgG. Out of 111 anti-HIV-1 positive specimens, collected mainly from Cameroonian HIV infected patients, only 10 were not reactive in this assay. The 42 anti-HIV-1 subtype O positive specimens gave all a reaction above cut off. Despite the diversity found in the amino acid sequences within the 25 isolates a peptide-based indirect ELISA representing the immunodominant epitope of the strain MVP-5180 successfully detected all the anti-HIV-O sera so far tested, pointing to the importance of adding such a peptide for correct identification of HIV-1 subtype O infected patients, while some assays without HIV-O specific antigens partially fail to detect all anti-HIV-O specimens.

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Reactivity of a new HIV-1 group O third generation A-HIV-1/-2 assay with an unusual HIV-1 seroconversion panel and HIV-1 group O/group M subtyped samples.

It was shown previously that about 97% of the anti-HIV-1 group O strain-positive samples were detected by crossreaction with native HIV-1 gp160 (Van Binsbergen et al., Evaluation of a new third generation anti-HIV-1/anti-HIV-2 assay with increased sensitivity for HIV-1 group O, J. Virol. Methods 60 (1996) 131-137). Fourteen out of 17 new anti-HIV-1 group O positive samples, selected with the Enzygnost HIV-1/2 plus assay, were already reactive when tested with HIV-1 gp160. When tested by the Vironostika HIV Uni-Form II plus O microELISA all 17 samples were reactive, demonstrating the necessity to implement an HIV-1 group O-specific antigen in the assay. On the other hand, it was surprisingly found that 40 out of 43 (93%) of anti-HIV-1 group M-positive samples, belonging to strain A, B, C, D, E or F, were detected by crossreaction with the HIV-1 group O (strain ANT70) synthetic peptide incorporated in the Vironostika HIV Uni-Form II plus O. Only HIV-1 subtype D-positive samples did not react with this peptide, presumably because of the presence of a histidine residue in the immunodominant region of HIV-1 subtype D gp41. Both crossreactions make the Vironostika HIV Uni-Form II plus O microELISA also sensitive for anti-HIV-1-positive samples originating from different geographical regions and resulting from different HIV-1 subtype infections. With an unusual seroconversion panel in which p24 Ag was present persistently, many anti-HIV-1/-2 assays produce alternating positive/negative results in anti-HIV antibody-positive bleeds. It was shown that the use of viral p24 and gp160 in a direct sandwich, allowing detection of anti-HIV IgG and IgM, explains the identification of all anti-HIV-positive bleeds by the Vironostika HIV Uni-Form II plus O. The high sensitivity of the plus O assay was confirmed with clinical samples of a so-called anti-HIV-1 low titer panel. The specificity of the Vironostika HIV Uni-Form II plus O determined in five blood transfusion centers, based on 135070 tests, was 99.97%.

Evaluation of a new third generation anti-HIV-1/anti-HIV-2 assay with increased sensitivity for HIV-1 group O.

Although the HIV-1 group O virus found in two persons of Cameroonian origin has been described in 1990 (De Leys et al., 1990), sera from group O infected individuals became available only recently. Several studies showed that some of the anti-HIV-1/HIV-2 screening tests failed to detect all of these samples (Loussert-Ajaka et al., 1994; Simon et al., 1994; Schable et al., 1994; Gürtler et al., 1995). In the current version of an anti-HIV-1/anti-HIV-2 screening assay, namely the Vironostika HIV Uni-Form II, an HIV-O specific peptide was introduced in order to improve HIV-1 group O reactivity. The peptide was derived from the immunodominant region of HIV-1 group O gp41 strain ANT70. All 30 anti-HIV-1 group O sera were detected by the so-called plus O assay, while 29 samples of this panel were positive the current assay. The sensitivity of the plus O assay for anti-HIV-1 and anti-HIV-2 positive samples is identical to that of the reference test without HIV-1 group O peptide. The clinical specificity of the HIV Uni-Form II plus O assay is improved to > or = 99.92% by an adjustment of the coat concentration of HIV-1 p24 (to avoid false positive p24 only reactions) without affecting sensitivity of the assay. The specific reaction of an HIV-1 group O specific rabbit serum for quality control purposes is presented.

HIV-1 subtype O: epidemiology, pathogenesis, diagnosis, and perspectives of the evolution of HIV.

HIV-1 subtype O is a new HIV variant originating in the West-Central African region, with highest prevalences in countries such as Cameroon, Equatorial Guinea and Gabon. Detection of antibodies to HIV-1 subtype O can pose problems in unmodified ELISA tests, and confirmation of anti-HIV-1 subtype O in immunoblot may give false negative results in some specimens. Nucleic acid-based assays designed for HIV-1 detection do not amplify or detect sequences from HIV-1 subtype O. In their env sequences, HIV-1 subtype O strains show a higher heterogeneity than the classical HIV-1 subtypes, leading to the conclusion that HIV-1 subtype O has been introduced into the human population only recently. Further, unidentified subtypes are also likely to exist.

Multicentre study for diagnostic evaluation of an assay for simultaneous detection of antibodies to HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0).

The aim of the study was to evaluate a new ELISA for detection of HIV-1, HIV-2 and HIV-1 subtype 0 (HIV-0) antibodies. The assay format is based on the antigen sandwich principle. To enable specific detection of HIV-0 antibodies, in addition to HIV-1 and HIV-2 antigens HIV-0 antigen is used for coating the solid phase and for the conjugate. The results show that all 12 HIV-0 samples tested were detected with a high degree of reactivity, as were all the 1,144 anti-HIV-1 and 424 anti-HIV-2 positive samples. The capacity of the test to enable early detection of seroconversions is equivalent to that of other sandwich ELISAs. The specificity of the assay was determined to be 99.89/99.94% (initial/after retest) using 58,366 samples, which is superior to the other ELISAs used for comparison. Even with difficult samples (i.e. samples of African origin, samples known to cause false-positive reactivity in different ELISAs, or samples containing potential interference factors) there were very few false-positive reactions. Therefore, the new assay is well suited for screening blood donations as well as for evaluating samples from patients of different geographic origin.

Reactivity of five anti-HIV-1 subtype O specimens with six different anti-HIV screening ELISAs and three immunoblots.

Five anti-subtype O specimens were tested by anti-HIV-1/2 screening and confirmatory assays. They can be divided into three specimens, reactive with all ELISAs, independent of the nature of the antigen (recombinant proteins or peptides) and test configuration (indirect ELISA or double antigen/sandwich ELISA). One specimen was not detected by one peptide based ELISA. One specimen was only recognized by two ELISAs and should be considered as a marker sample for the weakness of currently used ELISAs with anti-subtype O. Three different immunoblot assays available commercially detected two of the specimens with a major binding of gp160 and other viral bands, especially the integrase and reverse transcriptase. Another two specimens lacked reactivity with glycoproteins almost completely, but showed some staining with the enzymes of HIV, and would most probably be interpreted as indeterminate. The fifth specimen, which was also missed by most of the ELISAs, had very faint staining of the gp160 and a very weak staining of p24, and would most probably be interpreted as negative. Adaption of currently available tests to anti-subtype O is needed for the future reliability of anti-HIV diagnostic reagents.

Recombinant leech-derived tryptase inhibitor: construction, production, protein chemical characterization and inhibition of HIV-1 replication.

A synthetic gene coding for leech-derived tryptase inhibitor, form C (LDTI-C), was designed, cloned and expressed. The gene assembled via 6 oligonucleotides contains linker sequences, stop codons and internal restriction recognition sites for cloning, expression and cassette mutagenesis. Periplasmatic expression products could not be detected in Escherichia coli (E. coli), but strong expression was found using Saccharomyces cerevisiae (S. cerevisiae) ( > 10 mg/l culture broth) if a variant of pVT102U/alpha was used as vector. The secreted material was isolated after cross-flow filtration and purified by cation exchange chromatography. The recombinant material proved to be pure and homogeneous by electrophoretic and chromatographic analyses. Amino acid sequencing and molecular mass determination (4737.6 +/- 0.77 Da) by electrospray ionization mass spectrometry confirmed that rLDTI-C was processed correctly and that it is indistinguishable from LDTI-C. The far UV-CD (circular dichroism) spectrum of the recombinant inhibitor is typical for a small folded protein. rLDTI-C is inhibitorily fully active, its complexes with bovine trypsin and human mast cell tryptase display equilibrium dissociation constants which are nearly identical to those with the natural inhibitor. Remarkably, the inhibitor blocked replication of HIV-1 in HUT-78 cells at a concentration of 20 microM.

[Transfusion-associated HIV infection]. / Transfusionsassoziierte HIV-Infektion.

OBJECTIVE: Estimating the risk of HIV transmission by blood or inactivated plasma transfusion. A discussion of the methods and techniques for the diagnosis of HIV infection in blood donors. DATA SOURCES: Reports in German and English on this topic as well as own experiences of the authors. SELECTION CRITERIA: No specific selection criteria. RESULTS: Transfusion associated HIV infection may be prevented by donor selection, the very efficient anti-HIV testing, by p24-antigen testing that may detect some anti-HIV negative donations especially during the early time of seroconversion and by more recent introduced techniques like polymerase chain reaction (PCR) and signal amplification assay (SAA). PCR and SAA are under development and until now not sensitive and specific enough to contribute significantly to an earlier detection of HIV infected blood. Procedures for the inactivation of HIV in plasma or whole blood have been described. Until now use of psoralens, methylene blue or direct UV irradiation may reduce viral load but have not definitely been proven by clinical trials to be 100% efficient. CONCLUSIONS: To minimize transfusion associated HIV infection in future years reduction of total amount of transfused units, restriction to regional donor recruiting and further refinement of tests will be necessary. A 100% safety of blood transfusion for infectious agent cannot be achieved, especially considering new agents and further spread of until now geographically restricted viruses.

[HIV infection in ENT: risk of infection and preventive health precautions]. / HIV-Infektion in der HNO: Infektionsrisiko und Hygienevorschriften.

Risk of HIV transmission during the different modes of treatment depends on the HIV prevalence within the patient population and the instruments used during invasive procedures. Generally HIV may be transmitted from patient to the health care worker, from patient to patient and from health care worker to patient. Prevention of all modes of transmission is sterilisation or single use of equipment and if this is not possible proper disinfection of the equipment. HIV may be transmitted to patients by transplants that have not been sufficiently checked or pretreated. The most common route of HIV transmission is by stab wounds or puncture wounds by sharp instruments including needles. All reagents used for disinfection of other infectious agents are also valid for HIV. Infected staff should when ever possible avoid or refrain from performing operative procedures. HIV transmission is easily to prevent and part of all daily activities in medicine.

A new subtype of human immunodeficiency virus type 1 (MVP-5180) from Cameroon.

A new subtype (MVP-5180) of human immunodeficiency virus type 1 (HIV-1) was isolated from a Cameroonian AIDS patient. MVP-5180 was grown in several human T-cell lines and the monocytic U937 line. MVP-5180 DNA could not be amplified by nested primer PCR with conventional env primers and could be only very faintly amplified with gag and pol primers. Most German, Ivoirian, and Malawian anti-HIV-1 sera reacted faintly or moderately with Env proteins in an MVP-5180 immunoblot, whereas some Cameroonian sera reacted strongly. Of HIV-1-infected Cameroonians, 8% were identified by serological methods as infected with MVP-5180; 7% were positive when MVP-5180-specific PCR env primers were used. DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49%, respectively. V3 loop analysis identified a crown of Gly-Pro-Met-Arg by using cloned DNA and Gly-Pro-Leu-Arg by using PCR-amplified DNA, neither of which configuration has been described for other HIV strains. In an analysis of relationships, MVP-5180 occupied a position distant to all other HIV-1 strains, including the chimpanzee simian immunodeficiency virus type 1 SIVcpz and the Uganda virus U455, and closer to the HIV-1/HIV-2 divergence node. MVP-5180, together with another Cameroonian isolate, ANT-70, constitutes a group subtype O of the most divergent HIV-1 isolates yet identified. Characterization of MVP-5180 is important for understanding the natural history of the primate immunodeficiency viruses and for the development of vaccines and diagnostics.

Haptoglobin (HP), transferrin (TF) and group-specific component (GC) subtype distribution in Bantu-speaking people from Malawi.

A sample from Malawi was studied for the genetic markers haptoglobin (HP), group-specific component (GC) and transferrin (TF). The following allele frequencies were found. For HP: 1F = 0.355, 1S = 0.204, 2FS = 0.396, 2SS = 0.044; the allele 2FF was not observed. For GC: 1F = 0.814, 1S = 0.057, 2 = 0.079, 1A1 = 0.047, 2A1 = 0.0025. For TF: C1 = 0.894, C2 = 0.075, C3 = 0.0026, D1 = 0.029. The HP subtype distribution is among the first to be reported for African blacks.