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International audience; The Second Annual Meeting of the European Virus Bioinformatics Center (EVBC), held in Utrecht, Netherlands, focused on computational approaches in virology, with topics including (but not limited to) virus discovery, diagnostics, (meta-)genomics, modeling, epidemiology, molecular structure, evolution, and viral ecology. The goals of the Second Annual Meeting were threefold: (i) to bring together virologists and bioinformaticians from across the academic, industrial, professional, and training sectors to share best practice; (ii) to provide a meaningful and interactive scientific environment to promote discussion and collaboration between students, postdoctoral fellows, and both new and established investigators; (iii) to inspire and suggest new research directions and questions. Approximately 120 researchers from around the world attended the Second Annual Meeting of the EVBC this year, including 15 renowned international speakers. This report presents an overview of new developments and novel research findings that emerged during the meeting.

Contains fulltext : 154919.pdf (Publisher’s version ) (Closed access) OBJECTIVE: This study aimed to evaluate the effect of dendritic cell (DC) vaccination against HIV-1 on host gene expression profiles. DESIGN: Longitudinal PBMC samples were collected from participants of the DC-TRN trial for immunotherapy against HIV. Microarray-assisted gene expression profiling was performed to evaluate the effects of vaccination and subsequent interruption of antiretroviral therapy on host genome expression. Data from the DC-TRN trial were compared with results from other vaccination trials. METHODS: We used Affymetrix GeneChips for microarray gene expression analysis. Data were analyzed by principal component analysis and differential gene expression was assessed using linear modeling. Gene ontology enrichment and gene set analysis were used to characterize differentially expressed genes. Transcriptome analysis included comparison with PBMCs obtained from DC-vaccinated melanoma patients and of healthy individuals who received seasonal influenza vaccination. RESULTS: DC-TRN immunotherapy in HIV-infected individuals resulted in a major shift in the transcriptome. Longitudinal analysis demonstrated that changes in the transcriptome sustained also during interruption of antiretroviral therapy. After DC-vaccination, the transcriptome was enriched for cellular immunity associated genes that were also induced in healthy adults who received live attenuated influenza virus vaccination. These beneficial responses were accompanied by detrimental signals of general immune activation. CONCLUSIONS: The DC-TRN induced changes in the transcriptome were profound, lasting, and consisted of both protective signals and signatures of inflammation and immune exhaustion, with a net result of decreased viral load, without clinical benefit. Thus transcriptome analysis provides useful information, dissecting both positive and negative effects, for the evaluation of safety and efficacy of immunotherapeutic strategies.

Influenza virus infection is an important cause of severe asthma exacerbations, but it remains unclear how a Th1‐mediated antiviral response triggers a prototypical Th2 disease. We investigated CD4+ T cells and group 2 innate lymphoid cells (ILC2s) in influenza virus‐infected mice. We found that ILC2s accumulated in the lung rapidly after influenza virus infection, but the induction of IL‐5 and IL‐13 secretion was delayed and concomitant with T cell activation. In an influenza‐induced exacerbation of allergic airway inflammation model we noticed an initial reduction of ILC2 numbers and cytokine production in broncho‐alveolar lavage compared to chronic house dust mite (HDM)‐mediated airway inflammation alone. ILC2s phenotype was characterized by low T1/ST2, ICOS, KLRG1, and CD25 expression, resembling naïve ILC2s. The contribution of ILC2s to type 2 cytokine production in the early stage of the influenza‐induced exacerbation was limited. In contrast, T cells showed increased IL‐4 and IL‐5 production when exposed to both HDM and influenza virus. Upon virus clearance, ILC2s regained an activated T1/ST2highICOShighKLRG1highCD25high phenotype paired with cytokine production and were major contributors to the type 2 cytokine milieu. Collectively, our data indicate that both T cells and ILC2s contribute to influenza‐induced exacerbation of allergic airway inflammation, but with different kinetics.

Helper T cell differentiation is a key process in the regulation of adaptive immune responses. Here, mouse Th1 and Th2 cells are profiled using high-throughput proteomics to increase the understanding of the molecular biology of Th differentiation to support the design of prophylactic and therapeutic intervention strategies for (infectious) diseases. Protein profiling of Th1/Th2 differentiated cells results in the quantification of almost 6000 proteins of which 41 are differentially expressed at FDR < 0.1, and 19 at the FDR < 0.05 level, respectively. Differential protein expression analysis identifies a number of the expected canonical Th differentiation markers, and gene set analysis using the REACTOME database and a hypergeometric test (FDR < 0.05) confirms that helper T cell pathways are the top sets that are differentially expressed. Additionally, by network analysis, many differentially expressed proteins are associated with the Th1 and Th2 pathways. Data are available via PRIDE database with identifier PXD004532.

Th cells can adopt a number of different phenotypes. We performed microarray-assisted mRNA profiling on antigen-stimulated, TCR transgenic murine splenocytes that were cultured in the presence of cytokines. Transcriptome snapshots of Th cells differentiating into Th1 and Th2 phenotypes were obtained at various time points. Principal component analysis shows that time since activation and Th skewing are the largest sources of variance (i.e. the largest contributing factors) in our profiling experiments. Divergence between the Th1 and Th2 phenotypes is established early and does not increase in terms of number of differential genes from day 1 to day 4 after stimulation. Notwithstanding the lack of further divergence between the Th1 and Th2 lineages, we show that gene expression is best described by a 'turnover' rather than a 'core response' model, although we find evidence for both. We identify clusters of skewed genes associated with early persistent ('core response') and late ('turnover') Th1 and Th2 gene expression. In addition to the classical Th genes, members of the Batf transcription factor family are differentially expressed in particular helper phenotypes, suggesting an important role for this family in Th-cell phenotype differentiation.

The T helper paradigm is currently being revised from the Th1-Th2 dichotomy to a multi-state paradigm involving a number of different cell phenotypes. Transcriptional profiling using microarrays has been used to study the development of these phenotypes. There is however no clear consensus on how to approach the analysis of this data, especially in the context of cells that are triggered to expand rapidly, and massively change their gene expression pattern. We develop a method we call 'polar score' to identify genes that are related to T helper cell polarization. This method is designed to identify polarizing genes in a set where many genes change expression. To illustrate the use of this technique, we apply it to published T cell microarray data and compare it to conventional analysis methods. With the new method, we find evidence for the existence of 119 producing T cells (119 cells') that are induced by a combination of TGF beta and IL4. We identify several candidate master regulator genes for this phenotype. Furthermore, treatment with TGF beta and IL12 results in a Treg and Th17 hybrid cell phenotype. (C) 2010 Elsevier B.V. All rights reserved.

Contains fulltext : 89534.pdf (Publisher’s version ) (Closed access) Respiratory virus infections are among the primary causes of morbidity and mortality in humans. Influenza virus, respiratory syncytial virus (RSV), parainfluenza (PIV) and human metapneumovirus (hMPV) are major causes of respiratory illness in humans. Especially young children and the elderly are susceptible to infections with these viruses. In this study we aim to gain detailed insight into the molecular pathogenesis of respiratory virus infections by studying the protein expression profiles of infected lung epithelial cells. A549 cells were exposed to a set of respiratory viruses [RSV, hMPV, PIV and Measles virus (MV)] using both live and UV-inactivated virus preparations. Cells were harvested at different time points after infection and processed for proteomics analysis by 2-dimensional difference gel electrophoresis. Samples derived from infected cells were compared to mock-infected cells to identify proteins that are differentially expressed due to infection. We show that RSV, hMPV, PIV3, and MV induced similar core host responses and that mainly proteins involved in defense against ER stress and apoptosis were affected which points towards an induction of apoptosis upon infection. By 2-D DIGE analyses we have gathered information on the induction of apoptosis by respiratory viruses in A549 cells.

Genomics tools allow us to assess gene expression 'genome wide' providing an unprecedented view on the host-side of the virus-host interaction. The success of the application of these tools crucially depends on our ability to reduce the total information load while increasing the information density of the data collected. In addition to the advanced data analysis algorithms, gene an notation-pathway databases, and theoretical models, specifically designed sets of complementary experiments are crucial in translating the collected genomics data into palatable knowledge. A better understanding of the molecular basis of virus-host interactions will support the rational design of improved and novel intervention strategies for viral infections.

To identify immunological mechanisms that govern distinct clinical phases of a chronic hepatitis B virus (HBV) infection—immune tolerant (IT), immune active (IA), inactive carrier (IC), and hepatitis B e antigen (HBeAg)-negative (ENEG) hepatitis phases—we performed a systems biology study. Serum samples from untreated chronic HBV patients (n = 71) were used for multiplex cytokine measurements, quantitative hepatitis B surface antigen (HBsAg), HBeAg levels, HBV genotype, and mutant analysis. Leukocytes were phenotyped using multicolor flow cytometry, and whole-blood transcriptome profiles were generated. The latter were compared with liver biopsy transcriptomes from IA (n = 16) and IT (n = 3) patients. HBV viral load as well as HBeAg and HBsAg levels (P

Measles remains endemic in many East African countries, where it is often associated with high morbidity and mortality. We collected clinical specimens from Sudanese measles patients between July 1997 and July 2000. Sequencing of the 3′ 456 nucleotides of the nucleoprotein gene from 33 measles virus (MV) isolates and 8 RNA samples extracted from clinical specimens demonstrated the presence of a single endemic MV strain with little sequence variation over time (overall nucleotide divergence of 0 to 1·3%). This was confirmed by sequencing of the complete H gene of two isolates from 1997 and two from 2000, in which the overall divergence ranged between 0 and 0·5%. Comparison with MV reference strains demonstrated that the viruses belonged to clade B, genotype B3, and were most closely related to a set of viruses recently isolated in Nigeria. Our study demonstrates a remarkable genetic stability of an endemically circulating MV strain.

Respiratory syncytial virus (RSV) is a major cause of severe respiratory disease in infants and the elderly. RSV vaccine development has been hampered by results of clinical trials in the 1960s, when formalin-inactivated whole-RSV preparations adjuvated with alum (FI-RSV) were found to predispose infants for enhanced disease following subsequent natural RSV infection. We have reproduced this apparently immunopathological phenomenon in infant cynomolgus macaques and identified immunological and pathological correlates. Vaccination with FI-RSV induced specific virus-neutralizing antibody responses accompanied by strong lymphoproliferative responses. The vaccine-induced RSV-specific T cells predominantly produced the Th2 cytokines interleukin-13 (IL-13) and IL-5. Intratracheal challenge with a macaque-adapted wild-type RSV 3 months after the third vaccination elicited a hypersensitivity response associated with lung eosinophilia. The challenge resulted in a rapid boosting of IL-13-producing T cells in the FI-RSV-vaccinated animals but not in the FI-measles virus-vaccinated control animals. Two out of seven FI-RSV-vaccinated animals died 12 days after RSV challenge with pulmonary hyperinflation. Surprisingly, the lungs of these two animals did not show overt inflammatory lesions. However, upon vaccination the animals had shown the strongest lymphoproliferative responses associated with the most pronounced Th2 phenotype within their group. We hypothesize that an IL-13-associated asthma-like mechanism resulted in airway hyperreactivity in these animals. This nonhuman primate model will be an important tool to assess the safety of nonreplicating candidate RSV vaccines.

Respiratory syncytial virus (RSV) is the most important cause of bronchiolitis and pneumonia in infants and young children. Immunopathology may play a role in RSV-induced disease and a severe RSV infection may also be associated with an increased risk of developing asthma. Vaccination with formalin-inactivated RSV (FI-RSV) prior to infection resulted both in human and in the mouse model in extensive lung pathology. In the mouse model, it has been shown that this aggravation of disease was associated with a shift in the balance between Th1 and Th2 cytokines towards a Th2-type response. The aim of the present study was to characterise the immunological and inflammatory responses in BALB/c mice upon RSV infection with or without prior vaccination with aluminium-adjuvanted FI-RSV or control antigens (FI-Mock). As previously reported by others, we also observed that a primary RSV infection in BALB/c mice resulted in a predominant Th1-type cytokine response, which was associated with slight bronchiolitis and alveolitis. FI-RSV vaccination prior to RSV challenge prevented virus replication and was associated with an aggravation of pulmonary histopathology and a shift towards a Th2-type response. Vaccination with FI-Mock did not prevent RSV replication in the lung but resulted in an even more pronounced Th2 response after infection while these mice were not sensitised to specific viral antigens. Thus, viral replication in a Th2 responding animal (induced by aluminium-adjuvanted mock vaccine) appears to boost the Th2 response upon RSV infection.

Recently, we and others have shown that the interaction between envelope specific antibodies and primary human immunodeficiency virus type 1 (HIV-1) isolates may result in either inhibition or enhancement of virus entry. The outcome proved to be determined by the virus isolate rather than by the specificity of the antiserum used. To study the mechanism underlying this phenomenon, a series of HIV-1 envelope glycoproteins from closely related primary virus isolates of different syncytium inducing phenotypes, together with chimeras of these proteins, were tested in an envelope trans-complementation assay for their sensitivity to either antibody mediated inhibition or enhancement of HIV-1 entry. Based on the observation that, in contrast to the inhibition of HIV-1 entry, antibody mediated enhancement was not temperature dependent and could not be mediated by F(ab) fragments, we concluded that the mechanisms underlying these phenomena are different and that antibody mediated enhancement of HIV-1 entry is largely if not exclusively mediated by HIV-1 glycoprotein cross-linking. The susceptibility of the envelope glycoprotein chimeric viruses to neutralization or enhancement of infectivity proved to be primarily determined by the configuration of the V3 loop, and the affinity of the antibodies to monomeric HIV-1 gp 160 molecules, proved to be of quantitative importance only. One human monoclonal antibody directed against gp41 (IAM 2F5) inhibited entry of all the viruses studied, irrespective of their phenotype, and directly proportional to its affinity to monomeric HIV-1 gp 160.

Helper T (Th)-cell differentiation is a key event in the development of the adaptive immune response. By the production of a range of cytokines, Th cells determine the type of immune response that is raised against an invading pathogen. Th cells can adopt many different phenotypes, and Th-cell phenotype decision-making is crucial in mounting effective host responses. This review discusses the different Th-cell phenotypes that have been identified and how Th cells adopt a particular phenotype. The regulation of Th-cell phenotypes has been studied extensively using mathematical models, which have explored the role of regulatory mechanisms such as autocrine cytokine signalling and cross-inhibition between self-activating transcription factors. At the single cell level, Th responses tend to be heterogeneous, but corrections can be made soon after T-cell activation. Although pathogens and the innate immune system provide signals that direct the induction of Th-cell phenotypes, these instructive mechanisms could be easily subverted by pathogens. We discuss that a model of success-driven feedback would select the most appropriate phenotype for clearing a pathogen. Given the heterogeneity in the induction phase of the Th response, such a success-driven feedback loop would allow the selection of effective Th-cell phenotypes while terminating incorrect responses.

Background Severe dengue virus (DENV) disease is associated with extensive immune activation, characterized by a cytokine storm. Previously, elevated lipopolysaccharide (LPS) levels in dengue were found to correlate with clinical disease severity. In the present cross-sectional study we identified markers of microbial translocation and immune activation, which are associated with severe manifestations of DENV infection. Methods Serum samples from DENV-infected patients were collected during the outbreak in 2010 in the State of São Paulo, Brazil. Levels of LPS, lipopolysaccharide binding protein (LBP), soluble CD14 (sCD14) and IgM and IgG endotoxin core antibodies were determined by ELISA. Thirty cytokines were quantified using a multiplex luminex system. Patients were classified according to the 2009 WHO classification and the occurrence of plasma leakage/shock and hemorrhage. Moreover, a (non-supervised) cluster analysis based on the expression of the quantified cytokines was applied to identify groups of patients with similar cytokine profiles. Markers of microbial translocation were linked to groups with similar clinical disease severity and clusters with similar cytokine profiles. Results Cluster analysis indicated that LPS levels were significantly increased in patients with a profound pro-inflammatory cytokine profile. LBP and sCD14 showed significantly increased levels in patients with severe disease in the clinical classification and in patients with severe inflammation in the cluster analysis. With both the clinical classification and the cluster analysis, levels of IL-6, IL-8, sIL-2R, MCP-1, RANTES, HGF, G-CSF and EGF were associated with severe disease. Conclusions The present study provides evidence that both microbial translocation and extensive immune activation occur during severe DENV infection and may play an important role in the pathogenesis., Author Summary The pathogenesis of severe dengue virus (DENV) infection is still not fully understood. It is hypothesized that it is caused by a cytokine storm as is described in severe sepsis. In the sepsis field, the potent immunostimulator lipopolysaccharide (LPS) is proposed to play an important role in the development of a cytokine storm. In a previous study we have found elevated levels of LPS in children with severe DENV infection. In this study we have investigated if we could confirm that microbial translocation occurs in DENV-infected patients. Moreover, we have determined the levels of thirty cytokines to get more insight in the cytokine storm during DENV infections and we have investigated whether microbial translocation is associated with immune activation. The patients in this cohort were classified according to their clinical presentation. Furthermore, a cluster analysis based on the expression of the determined cytokines was applied to identify patients with similar cytokine profiles. With these two techniques, we identified cytokines that may contribute significantly to the cytokine storm, and we could relate elevated levels of LPS to patients with a pro-inflammatory cytokine profile.

Several studies have demonstrated a functional role for the V1-V2 region of the human immunodeficiency virus type 1 (HIV-1) envelope surface glycoprotein gp120 in the membrane fusion processes underlying viral entry and syncytium induction. In a study with chimeric primary envelope genes, we have previously demonstrated that the exchange of V2 regions was sufficient to transfer syncytium-inducing capacity to a non-syncytium-inducing envelope protein. The exchanged V2 regions, comprising a number of variable amino acids, conferred changes to both the predicted secondary structure and to the net positive charge of the V2 loops. In a syncytium-forming assay based on transient envelope protein expression in CD4+ SupT1 cells, we have extended this observation by mutating the variable positions of the V2 region to determine the relative contribution of individual amino acids to syncytium formation. It can be shown that simultaneous mutation of multiple amino acids is needed to interfere with the V2 region-determined syncytium-inducing phenotype. Single amino acid changes either influencing charge of predicted secondary structure of the V2 loop proved to be insufficient to abolish V2 region-controlled syncytium formation. This robust V2 organization may allow the virus to accumulate mutations, while retaining its biological phenotype.

Enhancement of virus infectivity after sCD4 treatment has been documented for SIVagm and HIV-2. It has been suggested that a similar phenomenon may play a role in HIV-1 infection. In the present study we have analysed biological activities of virus neutralizing polyclonal and monoclonal human antibodies and of sCD4, towards HIV-1 chimeras with envelope proteins derived from one donor, which display different biological phenotypes. The antibodies, which recognize the V3 and/or the CD4 binding domains of the glycoproteins of these viruses and also sCD4 showed different levels of virus neutralizing activity toward the syncytium inducing HIV-1 strains. In contrast, they all dramatically enhanced the infectivity of an HIV-1 chimera with an envelope glycoprotein displaying the non-syncytium-inducing phenotype. Given the relatively conserved nature of non-syncytium-inducing HIV-1 surface glycoproteins early after infection, these data suggest a major role for antibody mediated enhancement of virus infectivity in the early pathogenesis of HIV-1 infection.

Ganglia of monkeys with reactivated simian varicella virus (SVV) contained more CD8 than CD4 T cells around neurons. The abundance of CD8 T cells was greater less than 2 months after reactivation than that at later times and correlated with that of CXCL10 RNA but not with those of SVV protein or open reading frame 61 (ORF61) antisense RNA. CXCL10 RNA colocalized with T-cell clusters. After SVV reactivation, transient T-cell infiltration, possibly mediated by CXCL10, parallels varicella zoster virus (VZV) reactivation in humans.

Th cells can adopt a number of different phenotypes. We performed microarray-assisted mRNA profiling on antigen-stimulated, TCR transgenic murine splenocytes that were cultured in the presence of cytokines. Transcriptome snapshots of Th cells differentiating into Th1 and Th2 phenotypes were obtained at various time points. Principal component analysis shows that time since activation and Th skewing are the largest sources of variance (i.e. the largest contributing factors) in our profiling experiments. Divergence between the Th1 and Th2 phenotypes is established early and does not increase in terms of number of differential genes from day 1 to day 4 after stimulation. Notwithstanding the lack of further divergence between the Th1 and Th2 lineages, we show that gene expression is best described by a 'turnover' rather than a 'core response' model, although we find evidence for both. We identify clusters of skewed genes associated with early persistent ('core response') and late ('turnover') Th1 and Th2 gene expression. In addition to the classical Th genes, members of the Batf transcription factor family are differentially expressed in particular helper phenotypes, suggesting an important role for this family in Th-cell phenotype differentiation.

To map the regions of the external envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) involved in the process of membrane fusion, we determined the syncytium-inducing capacity of a panel of transiently expressed chimeric envelope genes. This panel was generated by exchanging gene fragments between four previously studied envelope genes that exhibited a high degree of sequence homology yet displayed marked differences in syncytium-inducing capacity when expressed by recombinant vaccinia virus. The results demonstrate that multiple regions of the HIV-1 envelope glycoproteins are involved in syncytium formation. Some fragments, most notably those containing the V2 or V3 region, can transfer syncytium-inducing capacity to envelope proteins previously not capable of inducing syncytia. Moreover, it is shown that such regions functionally interact with other envelope regions, especially one encompassing the V4 and V5 regions of gp120 or a region encompassing part of gp41, to exert their function in membrane fusion.

Interspecies transmission of pathogens may result in the emergence of new infectious diseases in humans as well as in domestic and wild animals. Genomics tools such as high-throughput sequencing, mRNA expression profiling, and microarray-based analysis of single nucleotide polymorphisms are providing unprecedented ways to analyze the diversity of the genomes of emerging pathogens as well as the molecular basis of the host response to them. By comparing and contrasting the outcomes of an emerging infection with those of closely related pathogens in different but related host species, we can further delineate the various host pathways determining the outcome of zoonotic transmission and adaptation to the newly invaded species. The ultimate challenge is to link pathogen and host genomics data with biological outcomes of zoonotic transmission and to translate the integrated data into novel intervention strategies that eventually will allow the effective control of newly emerging infectious diseases.

Background A Java™ application is presented, which compares large numbers (n > 100) of raw FTICR mass spectra from patients and controls. Two peptide profile matrices can be produced simultaneously, one with occurrences of peptide masses in samples and another with the intensity of common peak masses in all the measured samples, using the peak- and background intensities of the raw data. In latter way, more significantly differentially expressed peptides are found between groups than just using the presence or absence in samples of common peak masses. The software application is tested by searching angiogenesis related proteins in glioma by comparing laser capture micro dissected- and enzymatic by trypsin digested tissue sections. Results By hierarchical clustering of the presence-absence matrix, it appears that proteins, such as hemoglobin alpha and delta subunit, fibrinogen beta and gamma chain precursor, tubulin specific chaperone A, epidermal fatty acid binding protein, neutrophil gelatinase-associated lipocalin precursor, peptidyl tRNA hydrolase 2 mitochondrial precursor, placenta specific growth hormone, and zinc finger CCHC domain containing protein 13 are significantly different expressed in glioma vessels. The up-regulated proteins in the glioma vessels with respect to the normal vessels determined by the Wilcoxon-Mann-Whitney test on the intensity matrix are vimentin, glial fibrillary acidic protein, serum albumin precursor, annexin A5, alpha cardiac and beta actin, type I cytoskeletal 10 keratin, calcium binding protein p22, and desmin. Peptide masses of calcium binding protein p22, Cdc42 effector protein 3, fibronectin precursor, and myosin-9 are exclusively present in glioma vessels. Some peptide fragments of non-muscular myosin-9 at the C-terminus are strongly up-regulated in the glioma vessels with respect to the normal vessels. Conclusion The less rigorous than in general used commercial propriety software de-isotope algorithm results in more mono-isotopic peptide masses and consequently more proteins. Centroiding of peptide masses takes place by taking the average over more spectra in the profile matrix. Cytoskeleton proteins and proteins involved in the calcium signaling pathway seem to be most up-regulated in glioma vessels. The finding that peptides at the C-terminus of myosin-9 are up-regulated could be ascribed to splicing or fragmentation by proteases.

This paper describes the development and evaluation of a new nested reverse transcription (RT)-PCR for the detection of rhinovirus in clinical samples. The nucleotide sequences of the 5′ noncoding regions of 39 rhinoviruses were determined in order to map the most conserved subregions. We designed a set of rhinovirus-specific primers and probes directed to these subregions and developed a new nested RT-PCR. The new assay includes an optimal RNA extraction method and amplicon identification with probe hybridization to discriminate between rhinoviruses and the closely related enteroviruses. It proved to be highly sensitive and specific. When tested on a dilution series of cultured viruses, the new PCR protocol scored positive at 10- to 100-fold-higher dilutions than a previously used nested RT-PCR. When tested on a collection of clinical samples obtained from 1,070 acute respiratory disease patients who had consulted their general practitioners, the new assay demonstrated a rhinovirus in 24% of the specimens, including all culture-positive samples, whereas the previously used PCR assay or virus culture detected a rhinovirus in only 3.5 to 6% of the samples. This new assay should help determine the disease burden associated with rhinovirus infections.

A type-specific human immunodeficiency virus type 1 (HIV-1)-neutralizing human monoclonal antibody (HuMAb MN215) is described that reacts with the V3 domain of a number of subtype B virus strains. Pepscan analysis indicated that amino acids at both sides of the tip of the V3 loop were involved in the binding of HuMAb MN215. The minimum epitope in a V3 sequence, obtained from the donor from whom the cell line originated, was 9 amino acids long and proved to be located at the C-terminal side of the tip of the loop. In a replacement Pepscan analysis, individual amino acids of the V3 loop important for binding of HuMAb MN215 were identified. Amino acids at positions 15 (H), 16 (I), 17 (G) and 18 (P) were found to be essential for binding of the antibody, whereas changes at positions 19 of G to N, 20 of R to K and 23 of F to L, as well as the addition of a negative charge at the C terminus, improved binding. Thus, amino acids involved in the binding of HuMAb MN215 are primarily located within highly variable regions of the V3 loop. HuMAb MN215 showed a higher affinity for the V3 domain sequences and recombinant envelope glycoproteins derived from non-syncytium-inducing strains than for those derived from syncytium-inducing strains.

To study HIV-1 envelope-mediated syncytium formation we have amplified, cloned, expressed, and sequenced individual envelope genes from a set of eight biological HIV-1 clones. These clones were obtained from two patients and display either a syncytium-inducing (SI) or nonsyncytium-inducing (NSI) phenotype. Upon expression through recombinant vaccinia virus, individual envelope gene products display heterogeneous syncytium-inducing capacities which reflect the phenotype of the parental biological HIV-1 clones in all cases. For the eight biological HIV-1 clones presented here, variation of the envelope gene alone is sufficient to explain the observed variable syncytium-inducing capacity of the respective parental viruses. In addition we determined the complete nucleotide sequence of these envelope genes. The predicted amino acid sequence revealed a considerable amount of variation located mainly in the previously denominated variable regions. In various regions of envelope genes obtained from the same patient, phenotype associated amino acid variation was found. This phenotype associated amino acid variation however, is not conserved between the two sets of envelope genes derived from different patients. Four envelope sequences derived from clones obtained from one patient showed phenotype-associated amino acid variation in the fusion domain. Sequencing of 12 additional fusion domains revealed that this same variation is found in four additional clones. However, a functional test performed on recombinant vaccinia expressing mutant envelope genes showed that this observed fusion domain variation does not contribute to the variation in syncytium-inducing capacity of the envelope gene product.