Guido van der Groen

A Dual Infection/Competition Assay Shows a Correlation between Ex Vivo Human Immunodeficiency Virus Type 1 Fitness and Disease Progression

ABSTRACT This study was designed to examine the impact of human immunodeficiency virus type 1 (HIV-1) fitness on disease progression through the use of a dual competition/heteroduplex tracking assay (HTA). Despite numerous studies on the impact of HIV-1 diversity and HIV-specific immune response on disease progression, we still do not have a firm understanding of the long-term pathogenesis of this virus. Strong and early CD8-positive cytotoxic T-cell and CD4-positive T-helper cell responses directed toward HIV-infected cells appear to curb HIV pathogenesis. However, the rate at which the virus infects the CD4+ T-cell population and possibly destroys the HIV-specific immune response may also alter the rate of disease progression. For HIV-1 fitness studies, we established conditions for dual HIV-1 infections of peripheral blood mononuclear cells (PBMC) and a sensitive HTA to measure relative virus production. A pairwise comparison was then performed to estimate the relative fitness of various non-syncytium-inducing/CCR5-tropic (NSI/R5) and syncytium-inducing/CXCR4-tropic (SI/X4) HIV-1 isolates. Four HIV-1 strains (two NSI/R5 and two SI/X4) with moderate ex vivo fitness were then selected as controls and competed against primary HIV-1 isolates from an HIV-infected Belgian cohort. HIV-1 isolates from long-term survivors (LTS) were outcompeted by control strains and were significantly less fit than HIV-1 isolates from patients with accelerated progression to AIDS (PRO). In addition, NSI/R5 HIV-1 isolates from PRO overgrew control SI/X4 strains, suggesting that not all SI/X4 HIV-1 isolates replicate more efficiently than all NSI/R5 isolates. Finally, there were strong, independent correlations between viral load and the total relative fitness values of HIV-1 isolates from PRO (r = 0.84, P = 0.033) and LTS (r = 0.86, P = 0.028). Separation of the PRO and LTS plots suggest that HIV-1 fitness together with viral load may be a strong predictor for the rate of disease progression.

Isolation and characterization of a new chimpanzee lentivirus (simian immunodeficiency virus isolate cpz-ant) from a wild-captured chimpanzee

ConclusionsThis study shows that natural infection of wild-captured chimpanzees with an HIV-related virus may not be uncommon. The diversity of the two chimpanzee isolates, the different geographical origin and the absence of disease suggest that chimpanzees have not recently become SIVcpz-infected.

EVALUATION OF A CLINICAL CASE-DEFINITION OF ACQUIRED IMMUNODEFICIENCY SYNDROME IN AFRICA

A provisional clinical case-definition for acquired immunodeficiency syndrome (AIDS) developed by the World Health Organisation (WHO) for use in Africa was tested on 174 inpatients at Mama Yemo Hospital, Kinshasa, Zaire. In this hospital population with a 34% infection rate of human immunodeficiency virus (HIV), the clinical case-definition had a specificity of 90%, a sensitivity of 59%, and a predictive value of 74% for HIV seropositivity. These results support the use of the WHO clinical definition for AIDS in Africa. However, since HIV prevalence and disease expression vary, similar evaluations should be carried out in different regions.

Identification of an env G subtype and heterogeneity of HIV-1 strains in the Russian Federation and Belarus

ObjectiveTo identify HIV-1 envelope sequence subtypes in infected individuals from the Russian Federation and Belarus. PatientsA cohort of children infected after exposure to non-sterile needles during the 1988–1989 HIV-1 epidemic in southern Russia (n = 20) and HIV-1-seropositive individuals from Russia (n = 1) and Belarus (n = 7) infected via sexual transmission. MethodsDNA samples derived from peripheral blood mononuclear cells were analysed for their HIV-1 genotypes by the heteroduplex mobility assay (HMA). The 1.3 kilobase-pair env gene fragments encoding a portion of gp120 were amplified by nested polymerase chain reaction, cloned and sequenced. The env sequences derived from these patients were aligned and phylogenetic neighbour-joining and maximum parsimony-derived trees generated. ResultsThe env sequences derived from eight individuals infected in Russia and Belarus belong to subtype A (one), B (four), C (two), and D (one). Sequences derived from children, infected during parenteral manipulations in southern Russia, and one mother were closely related, but highly divergent, as a group, from all prototypic strains (genetic divergence, 17.2–22.9%). However, they clustered together with env sequences of the V1525 and LBV21–7 isolates from Gabon, recently described to be members of a new HIV-1 env subtype G. ConclusionExtensive heterogeneity of HIV-1 subtypes was evident in the Russian Federation and Belarus. Our data also support the existence of an HIV-1 env genetic subtype G, and such isolates are now apparently present on both the African and European continents. These variants were identified through V3 peptide enzyme-linked immunosorbent assay screening and subsequent HMA analysis. The combination of these techniques represents a model for screening HIV variants within a large population.

A Search for Ebola Virus in Animals in the Democratic Republic of the Congo and Cameroon: Ecologic, Virologic, and Serologic Surveys, 1979–1980

More than 30 years after the first outbreak of Marburg virus disease in Germany and Yugoslavia and 20 years after Ebola hemorrhagic fever first occurred in central Africa, the natural history of filoviruses remains unknown. In 1979 and 1980, animals in the Democratic Republic of the Congo and Cameroon were collected during the dry season near the site of the 1976 Ebola hemorrhagic fever epidemic. The study objectives were to identify local animals and search for evidence of Ebola virus in their tissues. A total of 1664 animals representing 117 species was collected, including >400 bats and 500 rodents. Vero and CV-1 cells and IFA and RIA were used for virus and antibody detection, respectively. No evidence of Ebola virus infection was found. This study was limited in time and animal collections and excluded insects and plants. Long-term, prospective, multidisciplinary comparative studies will yield more information than will repeat short forays on the ecology of filoviruses.

Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae

The task of international expert groups is to recommend the classification and naming of viruses. The International Committee on Taxonomy of Viruses Filoviridae Study Group and other experts have recently established an almost consistent classification and nomenclature for filoviruses. Here, further guidelines are suggested to include their natural genetic variants. First, this term is defined. Second, a template for full-length virus names (such as “Ebola virus H.sapiens-tc/COD/1995/Kikwit-9510621”) is proposed. These names contain information on the identity of the virus (e.g., Ebola virus), isolation host (e.g., members of the species Homo sapiens), sampling location (e.g., Democratic Republic of the Congo (COD)), sampling year, genetic variant (e.g., Kikwit), and isolate (e.g., 9510621). Suffixes are proposed for individual names that clarify whether a given genetic variant has been characterized based on passage zero material (-wt), has been passaged in tissue/cell culture (-tc), is known from consensus sequence fragments only (-frag), or does (most likely) not exist anymore (-hist). We suggest that these comprehensive names are to be used specifically in the methods section of publications. Suitable abbreviations, also proposed here, could then be used throughout the text, while the full names could be used again in phylograms, tables, or figures if the contained information aids the interpretation of presented data. The proposed system is very similar to the well-known influenzavirus nomenclature and the nomenclature recently proposed for rotaviruses. If applied consistently, it would considerably simplify retrieval of sequence data from electronic databases and be a first important step toward a viral genome annotation standard as sought by the National Center for Biotechnology Information (NCBI). Furthermore, adoption of this nomenclature would increase the general understanding of filovirus-related publications and presentations and improve figures such as phylograms, alignments, and diagrams. Most importantly, it would counter the increasing confusion in genetic variant naming due to the identification of ever more sequences through technological breakthroughs in high-throughput sequencing and environmental sampling.

Virus nomenclature below the species level: A standardized nomenclature for filovirus strains and variants rescued from cDNA

Specific alterations (mutations, deletions, insertions) of virus genomes are crucial for the functional characterization of their regulatory elements and their expression products, as well as a prerequisite for the creation of attenuated viruses that could serve as vaccine candidates. Virus genome tailoring can be performed either by using traditionally cloned genomes as starting materials, followed by site-directed mutagenesis, or by de novo synthesis of modified virus genomes or parts thereof. A systematic nomenclature for such recombinant viruses is necessary to set them apart from wild-type and laboratory-adapted viruses, and to improve communication and collaborations among researchers who may want to use recombinant viruses or create novel viruses based on them. A large group of filovirus experts has recently proposed nomenclatures for natural and laboratory animal-adapted filoviruses that aim to simplify the retrieval of sequence data from electronic databases. Here, this work is extended to include nomenclature for filoviruses obtained in the laboratory via reverse genetics systems. The previously developed template for natural filovirus genetic variant naming, (/)///-, is retained, but we propose to adapt the type of information added to each field for cDNA clone-derived filoviruses. For instance, the full-length designation of an Ebola virus Kikwit variant rescued from a plasmid developed at the US Centers for Disease Control and Prevention could be akin to “Ebola virus H.sapiens-rec/COD/1995/Kikwit-abc1” (with the suffix “rec” identifying the recombinant nature of the virus and “abc1” being a placeholder for any meaningful isolate designator). Such a full-length designation should be used in databases and the methods section of publications. Shortened designations (such as “EBOV H.sap/COD/95/Kik-abc1”) and abbreviations (such as “EBOV/Kik-abc1”) could be used in the remainder of the text, depending on how critical it is to convey information contained in the full-length name. “EBOV” would suffice if only one EBOV strain/variant/isolate is addressed.

Subset markers of CD8(+) cells and their relation to enhanced cytotoxic T-cell activity during human immunodeficiency virus infection

Using fresh whole blood or isolated lymphocytes, the activity ofin vivo generated cytotoxic T-lymphocytes (CTL) was measured as the OKT3-specific lysis of HL-60 targets, in a cross-sectional study of 53 HIV(+) patients. CTL activity in the entire HIV(+) group was two to three times higher than in HIV(−) controls, with WHO stage 3 (=pre-AIDS) patients showing the highest cytolytic function. The whole-blood CTL assay was validated and its practical and theoretical advantages are discussed. Within the CD8(+) cells, the number and proportion of the CD45RO(+) “memory” subset were significantly increased in HIV(+) subjects. The HLA-DR(+) subset rose most spectacularly in the asymptomatic stage of the infection, while the CD38(+) subset was the only one still significantly rising between the pre-AIDS and the AIDS stage. CTL activity was most closely correlated with T8 cells expressing the CD38 marker. In the context of CTL, CD38 thus seems to reflect activation rather than immaturity. Lymphocytes from HIV(+) subjects with a high OKT3-specific lytic capacity also destroyed normal lymphoblasts to a significant extent, pointing to their possible involvement in an autodestructive process. Our data thus suggest the importance of T8 cytolytic function and/or T8 subtyping in the immunopathogenesis and the prognosis of HIV infection.

Identification and characterization of sera from HIV-infected individuals with broad cross-neutralizing activity against Group M (env clade A-H) and Group O primary HIV-1 isolates.

A previous study on cross‐clade neutralization activity, identified three key isolates, MNlab (envB/gagB; X4 coreceptor), VI525 (envG/gagH, envA/gagA; R5X4) and CA9 (Group O; R5), that allowed discrimination of sera, likely or unlikely to neutralize primary HIV‐1 isolates belonging to Group M (env clades A–H) and Group O. The prognostic ability of these three isolates was verified by means of an external validation on a different and larger set of sera. A total of 79 different sera (66 HIV‐1, 10 HIV‐2, 1 HIV‐1+2 and 2 SIVcpz) were examined first for their capacity to neutralize the three key isolates, next sera were challenged against 12 other primary HIV‐1 isolates of Group M (env A–H) and 2 isolates of Group O. Sera that neutralized all three isolates with an ID50 titer of ≥1/40, also neutralized the 14 other primary isolates belonging to different genetic groups and clades. Sera that did not neutralize all three isolates did not exert broad cross‐neutralizing activity. The neutralizing activity was antibody‐mediated because it was absorbed and eluted from a Prot‐G column. Competition‐neutralization experiments using recombinant gp120 (HIV‐1 MNlab) reduced the neutralizing capacity, suggesting that the neutralizing antibodies were directed against the Env protein. Remarkably, the broad cross‐neutralization activity was found primarily in African female patients. In conclusion, this study confirms that three isolates are sufficient to allow identification of broad cross‐neutralizing sera. J. Med. Virol. 61:14–24, 2000.

Detection of diverse variants of human immunodeficiency virus-1 groups M, N, and O and simian immunodeficiency viruses from chimpanzees by using generic pol and env primer pairs.

Human immunodeficiency virus type 1 (HIV-1) infection of humans is the result of independent cross-species transmissions of simian immunodeficiency viruses (SIVcpz) from naturally infected chimpanzees (Pan troglodytes troglodytes) to man. To develop a polymerase chain reaction-based assay capable of detecting members of all major phylogenetic SIVcpz and HIV-1 lineages (groups M, N, and O), primer pairs in conserved pol and env regions were designed. Both primer sets amplified </=10 copies of selected group M reference clones (subtypes A-H), proviral DNA or RNA of group N (YBF30), and group O of HIV-1 and also amplified divergent SIVcpz from cultured isolates (SIVcpzGAB1 and SIVcpzANT), uncultured spleen tissue (SIVcpzUS), and plasma (SIVcpzANT and SIVcpzUS). Sequences of the 2 amplicons (445 bp for gp41 and 261 bp for integrase) are of sufficient length for phylogenetic analyses, allowing both group and subtype classifications of the human viruses. Finally, both primer pairs are highly sensitive (>99%) in amplifying viral sequences from plasma taken from patients infected with HIV-1 group M (n=226) and O (n=17) viruses.