Genomic heterogeneity of AIDS retroviral isolates from North America and Zaire

In an analysis of the genomic variation of AIDS retroviral isolates from patients living in New York, Alabama, and Zaire, restriction maps were constructed by using seven enzymes, each known to cleave the proviral DNA more than once, in conjunction with Southern blot analysis. The maps of LAV, HTLV-III, and ARV-2 as deduced from their published nucleotide sequences were included in this analysis. The results demonstrated that (i) several "signature" restriction sites were common to all isolates; (ii) with the exception of LAV and HTLV-III, the North American and European isolates were all different from one another and showed no geographical specificity; (iii) the African isolates as a group were more diverse than those from North America and Europe; and (iv) the genomic variability was concentrated within the env gene.     

Genomic diversity of human T-lymphotropic virus type III (HTLV-III)

The DNA genomes of human T-lymphotropic virus type III (HTLV-III) isolated from 18 individuals with AIDS or who were at risk for AIDS were evaluated for evidence of variation. Although all of the 18 viral DNA's hybridized throughout their entire genomes to a full-length cloned probe of the original HTLV-III isolate, each of the 18 isolates showed a different restriction enzyme pattern. The number of restriction site differences between isolates ranged from only 1 site in 23 to at least 16 sites in 31. No particular viral genotype was associated with a particular disease state and 2 of the 18 patients had evidence of concurrent infection by more than one viral genotype. Propagation of three different viral isolates in vitro for up to 9 months did not lead to detectable changes in their restriction patterns. These findings indicate that different isolates of HTLV-III comprise a spectrum of highly related but distinguishable viruses and have important implications regarding the pathogenicity of HTLV-III and attempts to develop effective diagnostic, therapeutic, and preventive measures for this virus.     

Isolation of a new human retrovirus from West African patients with AIDS

The etiological agent of AIDS, LAV/HTLV-III, is common in Central Africa but is not endemic in other areas of that continent. A novel human retrovirus, distinct from LAV/HTLV-III, has now been isolated from two AIDS patients from West Africa. Partial characterization of this virus revealed that it has biological and morphological properties very similar to LAV but that it differs in some of its antigenic components. Although the core antigens may share some common epitopes, the West African AIDS retrovirus and LAV differ substantially in their envelope glycoproteins. The envelope antigen of the West African virus can be recognized by serum from a macaque with simian AIDS infected by the simian retrovirus termed STLV-IIImac, suggesting that the West African AIDS virus may be more closely related to this simian virus than to LAV. Hybridization experiments with LAV subgenomic probes further established that this new retrovirus, here referred to as LAV-II, is distantly related to LAV and distinct from STLV-IIImac.     

New human T-lymphotropic retrovirus related to simian T-lymphotropic virus type III (STLV-IIIAGM)

This report describes serologic evidence for a virus similar to that known as simian T-lymphotropic virus type III of African Green monkeys (STLV-IIIAGM) infecting apparently healthy people in Senegal, West Africa, and the isolation of virus from these individuals. Serum samples from selected healthy West African people showed unusual serologic profiles when tested with antigens of HTLV-III/LAV, the etiologic agent of AIDS, and of STLV-IIIAGM. The samples reacted strongly with all of the major viral antigens of STLV-IIIAGM but showed variable or no reactivity with the major viral antigens of HTLV-III/LAV by radioimmunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A new human T-lymphotropic virus (HTLV-IV) isolated from these people was grown in vitro and shown to have retroviral type particles, growth characteristics, and major viral proteins similar to those of the STLV-III and HTLV-III/LAV group of retroviruses. The gp120/160, gp32, p64, p55, p53, p24, and p15 proteins precipitated were the same size as and reactive with STLV-IIIAGM proteins. The serologic data suggest that this virus shares more common epitopes with STLV-IIIAGM than with the prototype HTLV-III/LAV that infects people in the United States and Europe. Further study of this virus and of the origin of the HTLV-III/LAV group of viruses may expand our understanding of the human AIDS virus.     

A new HTLV-III/LAV protein encoded by a gene found in cytopathic retroviruses

The DNA of the HTLV-III/LAV group of retroviruses contains certain additional open reading frames that are not found in typical avian or mammalian retroviruses. The role of these sequences in encoding for gene products that may be related to pathogenesis remains to be resolved. An open reading frame whose 5' end overlaps with the pol gene, but is unrelated to the env gene, has been observed in HTLV-III/LAV and visna virus, both cytopathic mammalian retroviruses. Evidence presented here shows that this open reading frame is a bona fide coding sequence of HTLV-III/LAV and that its product, a protein with a molecular weight of 23,000, induces antibody production in the natural course of infection.     

A new HTLV-III/LAV encoded antigen detected by antibodies from AIDS patients

A newly identified protein from HTLV-III/LAV, the virus implicated as the etiologic agent of the acquired immune deficiency syndrome, was studied. This protein, which has a molecular weight of 27,000 (p27), was shown by amino acid sequencing to have a coding origin 3' to the env gene on the HTLV-III genome. The presence of antibodies to p27 in virus-exposed individuals indicated that this gene is functional in the natural host.     

The epidemiology of AIDS: current status and future prospects

The reported incidence of acquired immune deficiency syndrome (AIDS) continues to increase in countries throughout the world. On the basis of a polynomial model for extrapolation, the cumulative number of cases diagnosed and reported since 1981 in the United States is expected to double during the next year with over 12,000 additional cases projected to be diagnosed by July 1986. The annual incidence rates for single (never-married) men in Manhattan and San Francisco, intravenous drug users in New York City and New Jersey, and persons with hemophilia A ranged from 261 to 350 per 100,000 population during 1984. For single men aged 25 to 44 years in Manhattan and San Francisco, AIDS was the leading cause of premature mortality in 1984 as measured by years of potential life lost. Infection with HTLV-III/LAV is considerably more common than reported AIDS in high-risk populations and can persist at least for several years, so the presence of specific antibody should be considered presumptive evidence of current infection. The screening of donated blood and plasma for antibody to HTLV-III/LAV and use of safer clotting factor concentrates should greatly reduce HTLV-III/LAV transmission through blood and blood products. Most HTLV-III/LAV infections occur through sexual transmission, use of contaminated needles, and as a result of infected mothers passing the virus to newborns. Continued research commitment is needed to develop an HTLV-III/LAV vaccine and therapy for this infection. In the interim, widespread community efforts are needed to minimize transmission.     

Isolation of a new virus, HBLV, in patients with lymphoproliferative disorders

A novel human B-lymphotropic virus (HBLV) was isolated from the peripheral blood leukocytes of six individuals: two HTLV-III seropositive patients from the United States (one with AIDS-related lymphoma and one with dermatopathic lymphadenopathy), three HTLV-III seronegative patients from the United States (one with angioimmunoblastic lymphadenopathy, one with cutaneous T-cell lymphoma, and one with immunoblastic lymphoma), and one HTLV-III seronegative patient with acute lymphocytic leukemia from Jamaica. All six isolates were closely related by antigenic analysis, and sera from all six virus-positive patients reacted immunologically with each virus isolate. In contrast, only four sera from 220 randomly selected healthy donors and none from 12 AIDS patients without associated lymphoma were seropositive. The virus selectively infected freshly isolated human B cells and converted them into large, refractile mono- or binucleated cells with nuclear and cytoplasmic inclusion bodies. HBLV is morphologically similar to viruses of the herpesvirus family but is readily distinguishable from the known human and nonhuman primate herpesviruses by host range, in vitro biological effects, and antigenic features.     

The role of mononuclear phagocytes in HTLV-III/LAV infection

Cells with properties characteristic of mononuclear phagocytes were evaluated for infectivity with five different isolates of the AIDS virus, HTLV-III/LAV. Mononuclear phagocytes cultured from brain and lung tissues of AIDS patients harbored the virus. In vitro-infected macrophages from the peripheral blood, bone marrow, or cord blood of healthy donors produced large quantities of virus. Virus production persisted for at least 40 days and was not dependent on host cell proliferation. Giant multinucleated cells were frequently observed in the macrophage cultures and numerous virus particles, often located within vacuole-like structures, were present in infected cells. The different virus isolates were compared for their ability to infect macrophages and T cells. Isolates from lung- and brain-derived macrophages had a significantly higher ability to infect macrophages than T cells. In contrast, the prototype HTLV-III beta showed a 10,000-fold lower ability to infect macrophages than T cells and virus production was one-tenth that in macrophage cultures infected with other isolates, indicating that a particular variant of HTLV-III/LAV may have a preferential tropism for macrophages or T cells. These results suggest that mononuclear phagocytes may serve as primary targets for infection and agents for virus dissemination and that these virus-infected cells may play a role in the pathogenesis of the disease.     

陕西省avian-like H1N1猪流感病毒的遗传进化分析

【目的】监测陕西省猪流感的流行与传播情况,为猪流感研究提供参考。【方法】从生猪养殖场和屠宰场表观健康猪中随机采取猪鼻/气管拭子样本,经SPF鸡胚分离,对HA阳性样本进行猪流感病毒RT-PCR鉴定。测定分离株全基因序列,通过MEGA4构建基因进化树,并进行遗传变异分析。【结果】从500份样本中分离鉴定出6株H1N1流感病毒,病毒分离率为1.2%。获得6个核苷酸相似性为98.3%—99.1%的分离株全基因序列。遗传进化分析表明6个分离株都位于avain-like H1N1猪流感病毒谱系,与中国江苏,辽宁,山东和香港等地H1N1猪流感病毒株同源性达97%—99%。6个分离株的8个基因片段均在蛋白受体结合位点、潜在糖基化位点等处发生核苷酸变异,这将会影响猪流感病毒的抗原特性、宿主适应性和致病力。【结论】首次从西北地区表观健康猪中分离到H1N1猪流感病毒,并获得6个分离株的全基因序列。遗传进化分析表明,6个分离株为avain-like H1N1猪流感病毒, 且多处氨基酸位点发生变异。     

Hepatitis B virus DNA sequences in lymphoid cells from patients with AIDS and AIDS-related complex

A lymphotropic virus HTLV-III/LAV was recently identified as the etiologic agent of the acquired immune deficiency syndrome (AIDS). In a study of concomitant hepatitis B infections in patients with AIDS or the AIDS-related complex, DNA sequences of hepatitis B virus (HBV) were found in fresh and cultured lymphocytes from patients with AIDS even in the absence of conventional HBV serological markers. Furthermore, the restriction DNA pattern was consistent with the integration of the viral DNA. These results should prompt additional studies to reevaluate a possible role of HBV as a cofactor in AIDS in addition to the HTLV-III/LAV causal agent.     

Characterization of gp41 as the transmembrane protein coded by the HTLV-III/LAV envelope gene

Radiolabeled amino acid sequencing was used to characterize gp41, an antigen of HTLV-III/LAV, the virus believed to be the etiological agent of the acquired immune deficiency syndrome. This antigen is the one most commonly detected in immunoblot assays by sera of patients with AIDS or AIDS-related complex (ARC) and other individuals infected with HTLV-III/LAV. A mouse monoclonal antibody that was reactive with gp41 precipitated a 160-kilodalton protein (gp160) in addition to gp41, but did not precipitate a 120-kilodalton protein (gp120) from extracts of metabolically labeled cells producing HTLV-III. Extracts of infected cells that had been labeled with tritiated leucine or isoleucine were immunoprecipitated with the monoclonal antibody. The immunoprecipitates were fractionated by polyacrylamide gel electrophoresis and the p41 was eluted from the gel bands and subjected to amino-terminal radiolabeled amino acid sequencing by the semiautomated Edman degradation. Leucine residues occurred in cycles 7, 9, 12, 26, 33, and 34 among 40 cycles and isoleucine occurred in cycle 4 among 24 cycles analyzed. Comparison of the data with the deduced amino acid sequence of the env gene product of HTLV-III precisely placed gp41 in the COOH-terminal region of the env gene product. Gp160 is thus the primary env gene product and it is processed into gp120 and gp41.     

Replicative and cytopathic potential of HTLV-III/LAV with sor gene deletions

The genome of the human T-lymphotropic virus type III (HTLV-III/LAV) has the potential to encode at least three polypeptides in addition to those encoded by the gag, pol, and env genes. In this study, the product of the sor (short open reading frame) region, which overlaps the 3' end of the pol gene, was found to be a protein with a molecular weight of 23,000. An assay was developed for testing the ability of cloned HTLV-III proviruses to produce viruses cytopathic for T4+ lymphocytes. In the cell line used, C8166, neither the HTLV-III sor gene product nor the complete 3'-orf gene product were necessary for the replication or cytopathic effects of the HTLV-III.     

Binding of HTLV-III/LAV to T4+ T cells by a complex of the 110K viral protein and the T4 molecule

Human T-lymphotropic virus type III (HTLV-III) or lymphadenopathy-associated virus (LAV) is tropic for human T cells with the helper-inducer phenotype, as defined by reactivity with monoclonal antibodies specific for the T4 molecule. Treatment of T4+ T cells with monoclonal antibodies to T4 antigen blocks HTLV-III/LAV binding, syncytia formation, and infectivity. Thus, it has been inferred that the T4 molecule itself is a virus receptor. In the present studies, the surfaces of T4+ T cells were labeled radioactively, and then the cells were exposed to virus. After the cells were lysed, HTLV-III/LAV antibodies were found to precipitate a surface protein with a molecular weight of 58,000 (58K). By blocking and absorption experiments, this 58K protein was identified as the T4 molecule. No cell-surface structures other than the T4 molecule were involved in the antibody-antigen complex formation. Two monoclonal antibodies, each reactive with a separate epitope of the T4 molecule, were tested for their binding capacities in the presence of HTLV-III/LAV. When HTLV-III/LAV was bound to T4+ T cells, the virus blocked the binding of one of the monoclonal antibodies, T4A (OKT4A), but not of the other, T4 (OKT4). When HTLV-III/LAV was internally radiolabeled and bound to T4+ T cells which were then lysed, a viral glycoprotein of 110K (gp110) coprecipitated with the T4 molecule. The binding of gp110 to the T4 molecule may thus be a major factor in HTLV-III/LAV tropism and may prove useful in developing therapeutic or preventive measures for the acquired immune deficiency syndrome.     

Isolation of T-lymphotropic retrovirus related to HTLV-III/LAV from wild-caught African green monkeys

Present evidence suggests that the acquired immune deficiency syndrome (AIDS) emerged in Central Africa as a new disease in recent decades. This disease has recently approached epidemic proportions in many parts of the world. The etiologic agent of AIDS is believed to be the virus HTLV-III/LAV, which has been proposed as having originated from a recent simian-human transmission in Africa. This report describes the isolation of a designated STLV-IIIAGM retrovirus closely related to HTLV-III/LAV from seven healthy wild-caught African Green monkeys (Cercopithecus aethiops) that showed the presence of antibodies designated STLV-IIIAGM. In vitro growth characteristics, ultrastructural morphology, and major proteins of 160,000 kilodaltons (kD), 120 kD, 55 kD, and 24 kD are similar to and cross-reactive with the analogous antigens of HTLV-III/LAV. The use of these serologic markers in the detection of STLV-IIIAGM-infected monkeys may be important in assuring the continued safety of a variety of biologic reagents that are derived from these primate species. The existence of a retrovirus closely related to HTLV-III/LAV that naturally infects an African nonhuman primate in the apparent absence of disease may provide a unique model for the study of human AIDS and the development of an effective vaccine.     

Sequence homology and morphologic similarity of HTLV-III and visna virus, a pathogenic lentivirus

A study was conducted of the genetic relation between human T-cell lymphotropic retroviruses and visna virus. The human T-cell lymphotropic viruses include those associated with T-cell malignancies (HTLV-I and HTLV-II) as well as the etiologic agent of the acquired immune deficiency syndrome (HTLV-III). Visna virus, a slowly replicating and pathogenic but nononcogenic retrovirus of sheep, is a member of the subfamily Lentivirinae. Results obtained by molecular hybridization and heteroduplex analysis indicated that a greater extent of nucleotide sequence homology exists between HTLV-III and visna virus than between HTLV-III and any of the other viruses. The homology observed under conditions of low stringency spanned the entire genome, but was strongest in the gag/pol region. The morphogenesis and fine structure of HTLV-III and visna virus also demonstrated striking similarities. The data provide strong evidence for a close taxonomic and thus evolutionary relation between HTLV-III and the Lentivirinae subfamily.     

犬瘟热病毒甘肃株的分离和N蛋白基因遗传进化分析

采用Vero细胞从甘肃2008-2009年疑似犬瘟热感染病犬体内分离到8株病毒,通过RT-PCR方法扩增M蛋白基因初步鉴定所获得毒株均为犬瘟热病毒(CDV)。进一步通过RT-PCR方法扩增分离株的N蛋白基因,并进行序列分析。结果表明,8株分离株均为CDV,它们之间N蛋白基因的核苷酸同源性和推定的氨基酸同源性分别为98.4%~99.7%、98.3%~99.6%;8株CDV分离株的N蛋白基因和TN、A75/17相应序列的核苷酸同源性分别为98.5%~99.5%、97.0%~98.0%,推定的氨基酸同源性分别为98.3%~99.2%、98.7%~99.8%;8株CDV分离株N蛋白基因和疫苗株(Onderstepoort和Snyder Hill)的相应序列的核苷酸同源性和推定的氨基酸同源性分别为93.0%~94.0%、96.8%~98.3%。根据N蛋白基因所建立的遗传系统发育树可知这8株CDV分离株与TN的亲缘关系较近,而与疫苗株处于不同的分支上。说明从甘肃分离的CDV野毒株和TN由同一毒株演化而来,与疫苗株亲缘关系较远。     

Expression in Escherichia coli of open reading frame gene segments of HTLV-III

Human T-cell lymphotropic virus type III (HTLV-III), the causative agent of the acquired immune deficiency syndrome (AIDS), was recently isolated and its genomic structure analyzed by DNA cloning methods. In the studies reported here a combined cloning and expression system was used to identify HTLV-III encoded peptides that react immunologically with antibodies in sera from AIDS patients. Cloned HTLV-III DNA was sheared into approximately 500-base-pair fragments and inserted into an "open reading frame" expression vector, pMR100. The inserted DNA was expressed in Escherichia coli transformants as a polypeptide fused to the lambda CI protein at its amino terminus and to beta-galactosidase at its carboxyl terminus. Sera from AIDS patients containing antibodies to HTLV-III were then used to screen for immunoreactive fusion proteins. Twenty clones, each specifying a fusion protein strongly reactive with AIDS serum, were identified. DNA sequence analysis indicated that the HTLV-III fragments were derived from the open reading frame DNA segments corresponding to the gag and pol gene coding regions and also the large open reading frame region (env-lor) located near the 3' end of the viral genome.     

HTLV-III/LAV-neutralizing antibodies to an E. coli-produced fragment of the virus envelope

Immunization with either an Escherichia coli recombinant segment of the human T-cell lymphotropic virus (HTLV-III/LAV) envelope protein (gp 120) or with deglycosylated gp 120 envelope protein produced antibodies that neutralize HTLV-III/LAV infection in vitro. Virus neutralization titers of these antisera were equivalent to those obtained with purified native gp120 as immunogen. This localizes at least one class of neutralizing epitopes to the carboxyl-terminal half of the molecule. In addition, native gp120 prevented HTLV-III/LAV--mediated cell fusion, whereas the recombinant gp120 fragment did not. This shows that although glycosylation is not required for induction of neutralizing antibodies, it may be important for interaction with CD4, the virus receptor. A segment of the HTLV-III/LAV envelope produced in E. coli may be an important ingredient of a vaccine for acquired immune deficiency syndrome.