[Two horses with neurological symptoms: could this be equine botulism?]. / Twee paarden met neurologische verschijnselen: is botulisme in het spel?

Symptoms, diagnosis and therapy of equine botulism are discussed by the presentation of two detailed reports of horses with neurological symptoms and the results of laboratory investigations over the period 2003-2008 in the Netherlands. In addition a brief summary of the available literature is presented. Prevailing symptoms of botulism in horses include paralysis of the tongue, salvation, dysphagia and paresis and paralysis of the skeletal muscles, as well as signs of colic. Symptoms and prognosis vary with the amount of botulinum neurotoxin (BoNT) involved. For early clinical diagnosis of botulism thorough investigation of the facial nerves is important, for instance by the use of the 'Tongue Stress Test'. Laboratory results often remain negative, probably due to the sampling time, the high sensitivity of horses for botulinum neurotoxin or treatment with antitoxins. Most clinical cases in horses are caused by botulinum neurotoxin B (BoNT/B). For therapy to be successful antiserum needs to be administered in the earliest possible stage of the disease and this should be supported by symptomatic therapy. Botulism is a feed-related intoxication caused by either carcasses in the roughage or BoNT/B production after poor conservation of grass silage. This is the main source of botulism in horses due to the popularity of individually packed grass silage as feed for horses. As long as no vaccine is available in the Netherlands quality control of silage and haylage is strictly recommended in order to reduce the risk of botulism in horses.

Ear packing after ear surgery: is it really necessary?

OBJECTIVE: We question the need for packing of the ear canal after ear surgery. For several years, it had not been the first author's standard practice to use post-operative ear packing. During this period, few problems or complications had been encountered. SETTING: Tertiary referral, academic, paediatric hospital. MATERIALS AND METHODS: A retrospective review of all children who had undergone major ear surgery in our unit over the last year was carried out. These cases represented a full range of otological procedures. Post-operative complications and infections in the first six post-operative weeks were recorded. RESULTS: A total of 135 ears were operated upon in 107 patients ranging in age from 11 months to 19 years (mean 9.5 years). During this time period, eight children (7.5 per cent) developed a post-operative ear infection. No cases of tympanic or meatal granulations, problems with the tympanomeatal flap, or meatal stenosis were encountered. All infections were successfully managed with topical antibiotics. DISCUSSION: We conclude that packing after ear surgery may be safely abandoned. This would not only save valuable operating time, but would also obviate the need for pack removal, always a source of discomfort and anxiety. This is especially important in children, who may subsequently require a further general anaesthesia in order to remove the pack.

Analysis of structural and physiological profiles to assess the effects of Cu on biofilm microbial communities.

We investigated the effects of copper on the structure and physiology of freshwater biofilm microbial communities. For this purpose, biofilms that were grown during 4 weeks in a shallow, slightly polluted ditch were exposed, in aquaria in our laboratory, to a range of copper concentrations (0, 1, 3, and 10 microM). Denaturing gradient gel electrophoresis (DGGE) revealed changes in the bacterial community in all aquaria. The extent of change was related to the concentration of copper applied, indicating that copper directly or indirectly caused the effects. Concomitantly with these changes in structure, changes in the metabolic potential of the heterotrophic bacterial community were apparent from changes in substrate use profiles as assessed on Biolog plates. The structure of the phototrophic community also changed during the experiment, as observed by microscopic analysis in combination with DGGE analysis of eukaryotic microorganisms and cyanobacteria. However, the extent of community change, as observed by DGGE, was not significantly greater in the copper treatments than in the control. Yet microscopic analysis showed a development toward a greater proportion of cyanobacteria in the treatments with the highest copper concentrations. Furthermore, copper did affect the physiology of the phototrophic community, as evidenced by the fact that a decrease in photosynthetic capacity was detected in the treatment with the highest copper concentration. Therefore, we conclude that copper affected the physiology of the biofilm and had an effect on the structure of the communities composing this biofilm.

Contrasting bacterioplankton community composition and seasonal dynamics in two neighbouring hypertrophic freshwater lakes.

We characterized the bacterioplankton community and its seasonal dynamics in two neighbouring hypertrophic lakes by denaturing gradient gel electrophoresis (DGGE) analysis of short (193 bp) 16S ribosomal DNA polymerase chain reaction (PCR) products obtained with primers specific for the domain Bacteria. Lake Blankaart is turbid and has a high phytoplankton biomass and episodic cyanobacterial blooms, whereas biomanipulated Lake Visvijver is characterized by clearwater conditions and the establishment of a dense charophyte vegetation. Both lakes were dominated by bacterial groups commonly found in freshwater habitats (e.g. ACK4 cluster of Actinomycetes; ACK stands for clones isolated from the Adirondack mountain lakes). Yet, cluster analysis and principal components analysis (PCA) revealed that taxon composition of the bacterioplankton community of the two lakes differs substantially and consistently throughout the season. During the study year (1998), the bacterioplankton community of both lakes showed a distinct seasonal pattern. Lake Blankaart showed a clear differentiation between winter, spring, summer and autumn. In Lake Visvijver, summer samples differed greatly from spring, autumn and winter samples. We hypothesize that the contrasting bacterioplankton in the two neighbouring shallow lakes is determined largely by the presence or absence of macrophytes.

Changes in bacterial and eukaryotic community structure after mass lysis of filamentous cyanobacteria associated with viruses.

During an experiment in two laboratory-scale enclosures filled with lake water (130 liters each) we noticed the almost-complete lysis of the cyanobacterial population. Based on electron microscopic observations of viral particles inside cyanobacterial filaments and counts of virus-like particles, we concluded that a viral lysis of the filamentous cyanobacteria had taken place. Denaturing gradient gel electrophoresis (DGGE) of 16S ribosomal DNA fragments qualitatively monitored the removal of the cyanobacterial species from the community and the appearance of newly emerging bacterial species. The majority of these bacteria were related to the Cytophagales and actinomycetes, bacterial divisions known to contain species capable of degrading complex organic molecules. A few days after the cyanobacteria started to lyse, a rotifer species became dominant in the DGGE profile of the eukaryotic community. Since rotifers play an important role in the carbon transfer between the microbial loop and higher trophic levels, these observations confirm the role of viruses in channeling carbon through food webs. Multidimensional scaling analysis of the DGGE profiles showed large changes in the structures of both the bacterial and eukaryotic communities at the time of lysis. These changes were remarkably similar in the two enclosures, indicating that such community structure changes are not random but occur according to a fixed pattern. Our findings strongly support the idea that viruses can structure microbial communities.

Genetic diversity of the biofilm covering Montacuta ferruginosa (Mollusca, bivalvia) as evaluated by denaturing gradient gel electrophoresis analysis and cloning of PCR-amplified gene fragments coding for 16S rRNA.

The shell of the bivalve Montacuta ferruginosa, a symbiont living in the burrow of an echinoid, is covered with a rust-colored biofilm. This biofilm includes different morphotypes of bacteria that are encrusted with a mineral rich in ferric ion and phosphate. The aim of this research was to determine the genetic diversity and phylogenetic affiliation of the biofilm bacteria. Also, the possible roles of the microorganisms in the processes of mineral deposition within the biofilm, as well as their impact on the biology of the bivalve, were assessed by phenotypic inference. The genetic diversity was determined by denaturing gradient gel electrophoresis (DGGE) analysis of short (193-bp) 16S ribosomal DNA PCR products obtained with primers specific for the domain Bacteria. This analysis revealed a diverse consortium; 11 to 25 sequence types were detected depending on the method of DNA extraction used. Individual biofilms analyzed by using the same DNA extraction protocol did not produce identical DGGE profiles. However, different biofilms shared common bands, suggesting that similar bacteria can be found in different biofilms. The phylogenetic affiliations of the sequence types were determined by cloning and sequencing the 16S rRNA genes. Close relatives of the genera Pseudoalteromonas, Colwellia, and Oceanospirillum (members of the gamma-Proteobacteria lineage), as well as Flexibacter maritimus (a member of the Cytophaga-Flavobacter-Bacteroides lineage), were found in the biofilms. We inferred from the results that some of the biofilm bacteria could play a role in the mineral formation processes.

Nearly identical 16S rRNA sequences recovered from lakes in North America and Europe indicate the existence of clades of globally distributed freshwater bacteria.

We compared bacterial 16S ribosomal RNA gene sequences recovered from Lake Loosdrecht, the Netherlands, to reported sequences from lakes in Alaska and New York State. In each of the three lake systems, which differ in pH and trophic state, some sequence types were found without related sequences (sequence identity < 90%) in the data sets from the other two systems. Two sequences in the Actinomycetes and Verrucomicrobia radiations were more closely related to sequences from the New York lakes data set than to any other sequence in the global databases. However, the most striking similarities were found in the subdivisions alpha and beta of the Proteobacteria. In these subdivisions three different clusters of highly related bacteria were identified (97-100% sequence identity) that were represented in all three lake regions. The clusters contained no members other than freshwater bacteria. One cluster falls within a monophyletic aquatic supergroup that apparently diverged early in evolution into an exclusive freshwater cluster and an exclusive marine cluster, the so-called SAR11 cluster. The detection of these three bacterial clades in lakes distinguished by geographic distance as well as physical and chemical diversity suggests that these organisms are dispersed globally and that they possess unique functional capabilities enabling successful competition in a wide range of freshwater environments.

Fine specificity of antibody recognition may predict amino acid substitution in the third variable region of gp120 during HIV type 1 infection.

To investigate how human immunodeficiency virus type 1 (HIV-1) escapes from antibodies directed against the neutralization domain in the third variable region (V3) of gp120, we examined precisely which amino acid contributed to antibody binding. From six HIV-1-infected individuals, sequential sera were tested for antibody binding to individually designed peptide panels. Each individual panel contained all V3 domain sequences of cloned HIV-1 variants obtained at several time points from the studied individual. We showed that the V3 domain is a major site for escape of the humoral immune response. We showed antibody binding was reduced by certain mutations in the V3 domain and sometimes concerted mutations rendered very distinct antigenic variants. The position and the number of the mutations that occurred during infection corresponded with the position and number of amino acids in the V3 domain that were important for binding to anti-V3 antibodies in the early immune response. The specificity of the antibody binding hardly changed during infection. Although mutations at several positions of the V3 domain reduced antibody binding, the mutations were limited to certain positions, probably because the function of the region has to be maintained. The amino acids that were important for binding in combination with the preference for changes at certain positions predicted to some extent the mutations that occurred later during infection.

Validation of allele-specific polymerase chain reaction for DNA typing of HLA-B27.

To find a specific method for HLA-B27 typing for the diagnosis of rheumatic disorders, we extensively tested the single-step B27-specific polymerase chain reaction (PCR) described by Dominguez et al. (Immunogenetics 1992;36:277-82). This method, which relies on specific primer recognition of a sequence in the third exon (unique to the B27-allele), was used for screening of 270 characterized blood samples, 57 of which were B27-positive. The method proved to be both sensitive and specific: It unambiguously identified all B27-positive samples and produced no false-positive results. For approximately 1% of the samples, we had to repeat DNA isolation and PCR to obtain a clear control amplification signal. In contrast to the specificity of the PCR method, parallel-performed flow cytometry gave ambiguous results in 3% of the samples because of antibody cross-reactivity. Flow cytometry and the PCR method described were similar in labor and costs. Therefore, we conclude that the proposed single-step PCR is feasible in a routine laboratory and would improve the reliability of HLA-B27 typing.

Antibody responses to HIV-1 envelope and gag epitopes in HIV-1 seroconverters with rapid versus slow disease progression.

We studied the relationship between the rate of disease progression after HIV-1 seroconversion and the level of IgG antibody response to HIV-1 envelope and core epitopes. This was done by comparing a group of fast-progressing individuals and a group of slow-progressing individuals for serum IgG titers to peptides from the gp120-V3 neutralization domain, to a peptide from the immunodominant gp41 epitope (residues 590 to 607), and to recombinant gp120 and p24. The two groups displayed a large overlap in titers to the envelope epitopes, which precluded their differentiation at most time points after seroconversion. Low responsiveness to envelope antigens was not only found in a few fast-progressors but also in one individual who remained asymptomatic for at least 92 months after seroconversion. The only significant differences between the groups were found in the first months after seroconversion when the responses to the V3 domain and the gp41 epitope were more vigorous in the group of fast-progressors. Furthermore, on evaluating ratios of anti-V3 antibody titers to anti-gp120 antibody titers we found no indication that fast disease progression was associated with a restriction in antibody response to the V3 epitope. We did confirm the finding that fast disease progression is associated with low levels of p24-directed antibodies, both early after seroconversion and at later stages. These data demonstrate that levels of IgG antibodies to envelope epitopes are poor predictors of rapid disease progression and suggest that the role of V3-directed neutralizing antibodies in preventing subversion of the immune system is not decisive in natural HIV-1 infection.

Frequent and early HIV-1MN neutralizing capacity in sera from Dutch HIV-1 seroconverters is related to antibody reactivity to peptides from the gp120 V3 domain.

The temporal development of HIV-1 neutralizing activity and antibodies to the gp120-V3 neutralization domain were studied in sera from 20 Dutch HIV-1-infected individuals followed from seroconversion on. Serum neutralizing capacity was assessed with three T cell line-tropic isolates: HIV-1MN, HIV-1HXB2, and the patient isolate HIV-1(320). Neutralizing activity to HIV-1MN developed in 18 individuals (90%) within 0 to 10 months after seroconversion. Parallel evolution of IgG reactivity to V3 peptides of United States/European type variants, and the capability of such peptides to completely inhibit HIV-1MN neutralization in four of five tested sera (taken 1-2 years after seroconversion), indicate that a large proportion of HIV-1MN neutralizing antibodies is directed to V3. The early appearance and high frequency of HIV-1MN neutralizing activity in the Dutch study group indicate the close relationship of HIV-1MN to HIV-1 variants circulating in the Netherlands. Neutralizing activity to HIV-1HXB2 (in 15 of 20 individuals) developed several months after that to HIV-1MN in all individuals (average, 10 months after seroconversion) and was not seen in the absence of HIV-1MN neutralizing activity. Neutralizing activity to the Dutch isolate HIV-1(320) (found in 11 of 18 tested individuals) emerged simultaneously with that to HIV-1MN in 4 individuals but appeared later in 7. In most individuals, HIV-1HXB2 neutralization was not accompanied by reactivity to a V3 peptide from this strain, indicating that the extension of neutralizing activity to more divergent strains, which takes place at later stages, must be attributed to non-V3-directed antibodies.

Increasing antigenic and genetic diversity of the V3 variable domain of the human immunodeficiency virus envelope protein in the course of the AIDS epidemic.

Population-wide variation in genomic RNA of human immunodeficiency virus type 1 (HIV-1) encompassing the V3 loop of the envelope protein was studied in serum samples of 74 newly infected individuals from three Dutch cohorts: 30 homosexual men, 32 drug users, and 12 hemophiliacs. During acute infection, HIV-1 RNA sequences present in serum are relatively homogeneous, which makes direct sequencing feasible. This offered an opportunity to study the infecting virus variants before mutations had accumulated in the new host. The sampling dates ranged from 1980 to 1991, thus spanning the entire AIDS epidemic in The Netherlands. The diversity in the sequenced region increased over time in both the homosexual and the drug-user risk groups. Furthermore, this increase was associated with an increase in antigenic variation, as witnessed by serum reactivity to a V3 peptide panel. Despite this diversification, some 1990 sequences still closely resembled the earliest 1980 sequence, making ancestral inferences problematic. No evidence was found of a change in the master sequence of the virus quasi-species over time. At the amino acid level, no risk-group-associated variation was found, but at the nucleotide level, the drug-user and homosexual/hemophiliac sequences could be distinguished on the basis of a single silent nucleotide change in the sequence encoding the tip of the V3 loop. Hemophiliac sequences could not be distinguished from those of homosexuals. In spite of the large and increasing genetic variability, all sequences were more similar to the European/American HIV consensus sequence than to that of non-Western strains.

Greater diversity of the HIV-1 V3 neutralization domain in Tanzania compared with The Netherlands: serological and genetic analysis.

OBJECTIVE: To compare variation in the HIV-1 V3 neutralization domain in Tanzania and The Netherlands. METHODS: For serologic analysis, the specificity of anti-V3 antibodies (immunoglobulin G) for a panel of V3 peptides was determined in sera from 55 symptomatic HIV-1-infected Tanzanians and 51 Dutch AIDS patients. For genetic analysis, viral RNA was isolated from 15 of the Tanzanian sera and six of the Dutch sera. The V3 encoding region was reverse-transcribed, polymerase chain reaction-amplified and bacterially cloned, and sequences were determined over a stretch of at least 207 nucleotides. RESULTS: Thirty-five per cent of the Tanzanian sera, versus 2% of the Dutch sera, showed the highest reactivity to a V3 sequence of Zairian origin (RKSIHVGPGQAFYATG). Twenty-nine per cent of the Tanzanian sera, versus 82% of the Dutch sera, showed the highest reactivity to V3 sequences of US/European origin (RKSIXIGPGRAFYTTG; X = H, P or N). The Tanzanian RNA sequences showed greater diversity (mean distance, 19%) than the Dutch RNA sequences (10%). The measured anti-V3 specificities of the Tanzanian sera did not match accurately with the V3 sequences recovered from these sera. However, reactivity to the Zairian V3 peptide was associated with the sequence GPGQ, found in the centre of the V3 in 50% of the Tanzanian sequences. Sera from both countries that showed similar reactivities to the peptide panel contained RNA sequences that were relatively distant. CONCLUSIONS: The diversity of the HIV-1 population in Tanzania is much greater than that in The Netherlands. An indication of the HIV-1 V3 variation in a particular geographic region can be obtained by serological methods, but sequence analysis should not be omitted.

Characterization of the specificity of the human antibody response to the V3 neutralization domain of HIV-1.

The major neutralization domain of HIV-1, contained in the third variable region (V3) of the external envelope, is highly variable at positions flanking a conserved glycine-proline-glycine sequence. We investigated the relation between V3 sequences of HIV-1 variants circulating in a host and that host's antibody specificity. Multiple V3 sequences were obtained directly, via PCR and subsequent cloning, from serum RNA or cellular DNA from 26 individuals (from 12 around seroconversion). Then, specificity of sera from these individuals to a panel of V3 peptides was determined. The specificity (best recognized peptide) of the early antibody response accurately reflected the virus population circulating around seroconversion in 12/12 individuals and 4/4 HIV-1-infected chimpanzees. A change in serum specificity at later stages of infection was rare: five years after seroconversion, only 3 of 46 individuals had a specificity that differed completely from that in the first year. However, the V3 domain of the virus does change over time, as evidenced by the poor correlation between V3 sequences obtained late in infection and V3 antibody reactivity at the same time point. Thus, in contrast to the accurate antibody response to HIV-1 variants early after infection, generally a specific response to variants emerging at later stages seemed to be absent or of low level. Instead, the early response appeared to be preserved. Finally, we made use of the observed accurate reflection to analyze the variation for the V3 domain of HIV-1 in the Netherlands by probing specificities of early sera from 129 Dutch seroconverting individuals. Specific reactivity to RKSIHIGPGRAFYTTG was found in 36%, to RKSINIGPGRAFYTTG in 12% and to RKSIPIGPGRAFYTTG in 18% of these Dutch sera.

HIV-1 genomic RNA diversification following sexual and parenteral virus transmission.

Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by epidemiological history and taken within a time bracket of three months of the recipient seroconversion, were analyzed by polymerase chain reaction amplification followed by sequencing of eight cDNA clones of 276 bp, including the V3 coding region. The sequence populations of the recipients were without exception homogeneous, while the sequence populations of the transmitters showed varying degrees of heterogeneity. Nucleotide distance between consensus sequences of unrelated individuals from the Amsterdam population (interpatient variation) averaged 11% (range 7-15%). The largest distance between two clonal sequences of one individual (intrapatient variation) was also 11%. Consensus sequences of five recipients differed by only 0-1% from the consensus sequence of the presumed transmitter, including two pairs of which the transmission was either proven or highly probable. This contrasted with a difference of 10-12% in two pairs, casting doubt on the epidemiological relatedness. Antibody reactivity to a panel of V3 peptides with varying degrees of similarity to the V3 sequences obtained did not augment the discriminatory power of sequence analysis. Results of the sequential sequencing of samples of one transmitter suggest that this was due to an anamnestic antibody response of the transmitter to early variants. From the loss of sequence heterogeneity following transmission and the consensus sequence similarities observed within five transmitter-recipient pairs, we conclude that HIV-1 transmission results in the selection of a limited number of genomes carrying on the infection in the new host, but does not generally lead to a shift in the sequence population as defined by the consensus sequence.

Naturally occurring mutations within HIV-1 V3 genomic RNA lead to antigenic variation dependent on a single amino acid substitution.

In a study on the evolution of genomic diversity of HIV-1, genomic RNA was isolated from serum of two individuals. Starting at the time of primary infection we collected six samples of serum from each patient over a period of 5 years. Ninety-four cDNA clones (50 of patient 1 and 44 of patient 495) of part of the envelope coding region including the principal neutralization domain (PND) were sequenced. Around the time of antibody seroconversion, genomic RNA levels reached a peak and the population of sequences was highly homogeneous. In the course of the infection, the number of amino acid substitutions accumulated, which led to a higher genomic diversity within successive samples and a drift in the consensus sequence, progressively differing from the first found consensus sequence. Fixation of a substitution at glycoprotein 120 amino acid 308 was observed in both patients between two time points (patient 1, H----P; patient 495, P----H). With the use of 16-meric synthetic peptides, differing only at the 308 position (H308 versus P308), antibody binding specificity was found to be dependent on this difference. In patient 495, the nonconservative (P308----H) substitution reduced the binding affinity with the patient's antibodies. Furthermore, antibody competition assays showed that the observed substitution at position 308 elicited a new antibody population, indicating antigenic variation. After the decline of V3-specific antibodies, the simultaneous increase in genomic RNA levels and progression to AIDS in patient 495, a new variant with major changes in the PND emerged, again forming a homogeneous population of sequences.

Immunodominance and antigenic variation of the principal neutralization domain of HIV-1.

The principal neutralization domain (PND) of HIV-1 is located in the third variable region (V3) of the envelope glycoprotein gp 120. Cross-reactivity of experimental and natural sera with recombinant proteins containing the V3 region of four HIV-1 variants showed that a group of viruses (among which HIV-1 MN) had antigenically similar V3 regions. The V3 regions of HIV-1 IIIB and HIV-1 RF were antigenically distinct. Antibodies raised to V3 domains of two isolates from the "MN group" neutralized HIV-1 MN (and not HIV-1 IIIB), thus confirming the antigenic similarity of V3 of these isolates to that of HIV-1 MN. Human antibodies to the V3 region were shown to be mainly directed to the central area in V3, where the neutralization domain is. In addition, antibody reactivities in sera of 397 Dutch and 39 Tanzanian HIV-1-infected individuals show that the V3 neutralization domain is highly antigenic, and that viruses from the MN group predominate in The Netherlands and to a lesser extent in Tanzania. Thus, if the protective value of antibodies to the PND can be established, then PND (poly)peptides derived from the MN group may be important components of a subunit vaccine against HIV-1.

Antibody recognition of amino acid divergence within an HIV-1 neutralization epitope.

Antibodies elicited by HIV-1 strains, and which neutralize such strains in vitro, bind to synthetic peptides of 5-8 amino acids in length. These amino acids, although variable, have a fixed location between two cysteines in the carboxyl terminus of the HIV-1 external envelope. Nine peptides of 9 amino acids corresponding to the gp120 domains of European and American (LAV-1, NY5, CDC4, SF2), Haitian (RF) and African (ELI, MAL, Z3, Z6) HIV-1 strains, were synthesized using LAV-1 and RF neutralization epitopes as models. Serum of chimpanzees infected with LAV-1, HTLV-IIIB or HTLV-IIIRF reacted predominantly with the homologous peptide, although cross-reactivity with heterologous peptides occurred: 8 out of 11 human sera with HTLV-IIIB-neutralizing activity bound the LAV-1/HTLV-IIIB peptide, and 6 out of 7 sera with HTLV-IIIRF-neutralizing activity bound the RF peptide. African sera reacted most frequently with the Z3 peptide (78%) while only 35% (p = 0.0001) of European and 20% (p less than 0.0001) of American sera recognized it. Recognition patterns of children from the USA and Europe were different. Although multiple reactivities were observed, blocking experiments favoured cross-reactivity as the explanation. Based on the antibody profiles of nonapeptide recognition, peptides LAV-1, RF and SF2 were clustered, as were NY5 and CDC4, and so were Z6, MAL and ELI. This antigenic relatedness of HIV-1 strains could not entirely be explained by the physico-chemical characteristics of the nonamers per se. Resemblance was observed with the clustering of HIV-1 strains based on the divergence of the nucleotide sequence of entire HIV-1 envelopes. This implies a role of peripheral envelope residues, in the context of infectious particles or infected cells, in determining the specificity of antibodies reactive to the V3 domain. Therefore, the neutralization domain in this variable region may be considered part of a conformational structure involving several envelope regions which appear distinct from each other in the primary sequence.