Analysis of partial gag and env gene sequences of HIV type 1 strains from southern Africa.

PIP: HIV-1 is spreading at an exponential rate in southern Africa, with a current doubling time of approximately one year. An estimated 2 million of South Africa's 36 million population are already infected with HIV. Information on the extent of variability of HIV-1 sequences in the region is important for the development of vaccines, the evaluation of new therapies, and for structure/function studies of the viral genome and proteins. The authors isolated and partially sequenced local strains of the virus. The first strain sequenced was determined to be a new subtype of HIV-1, designated subtype C(2). HIV-1 subtypes B and D are also circulating within southern Africa. The derived phylogenetic trees for the various strains are presented. It is possible that southern African HIV-1 strains have evolved from Central African ones during their spread southward over time and geographic distance. The data on HIV-1 env and gag gene variability presented in this paper have implications for the design of vaccines intended for use in southern Africa and India. The results also establish new limits of variability for the virus, by extending the phylogenetic tree along a new branch.^ieng

HIV-1 strains from India are highly divergent from prototypic African and US/European strains, but are linked to a South African isolate.

OBJECTIVE: To gain molecular insights into different HIV-1 strains present in two different states of India, nucleotide sequences derived from the env region of four HIV-1 strains were analysed. DESIGN: HIV-1 was isolated from high-risk patients from the states of Maharashtra (city of Bombay) and Goa. The molecular analysis of the env region encompassed all variable domains of the external glycoprotein, gp120. METHODS: Genomic DNA from cultured cells infected with each of the four Indian HIV-1 strains independently was amplified by polymerase chain reaction (PCR). PCR fragments were cloned and sequenced and a phylogenetic tree constructed. RESULTS: All four Indian HIV-1 sequences were closely related to each other. The closest related sequence to them was from a South African isolate, HIV-1NOF, with a homology of 85-87%. In the phylogenetic tree, the Indian and the South African HIV-1 sequences cluster together and constitute a subtype different from the North American/European, Central African, Uganda/Rwanda and Northern Thailand subtypes. Interestingly, the viruses of this subtype are characterized by an additional potential N-glycosylation site C-terminal to the CD4-binding domain. CONCLUSION: The low variation between the HIV-1 sequences from randomly chosen individuals from high-risk cohorts in two Indian states suggests a rapid and recent spread of HIV and, possibly, introduction of the virus by the same route, most probably heterosexual transmission. The rapid spread of HIV-1 variants in India, which form a subgroup of their own together with a South African strain, necessitate consideration of these strains in vaccine development.

Myelopathy associated with human T cell lymphotropic virus type I (HTLV-I) in natal, South Africa. A clinical and investigative study in 24 patients.

Unexplained spastic myelopathy in black (Zulu) patients, similar to that seen in the tropics, has previously been described from Natal, South Africa. Following reports linking the human T cell lymphotropic virus type I (HTLV-I) to spastic myelopathy, we undertook a prospective and retrospective search for HTLV-I antibodies in 36 patients who were labelled as having unexplained myelopathy; 24 (66%) were positive and HTLV-I was isolated from 4 out of the 6 patients whose peripheral blood lymphocytes were cultured. Eighteen (75%) gave a short history (less than 6 months). There was a female preponderance (71%), spinothalamic dysfunction was common (55%) and as many as half were severely disabled (50% wheelchair bound). Routine laboratory studies showed no specific trends apart from hypergammaglobulinaemia and CSF pleocytosis (greater than 5 cells/microliter in 66% of patients). The total CSF protein was raised (greater than 0.4 g/l) in 45% of patients. The IgG index was greater than 0.7 in 15 of 19 patients. Conventional myelography did not show any specific abnormalities. Computer assisted myelography was undertaken in 22 patients; 3 showed arachnoiditis and 2 spinal cord atrophy. Periventricular lucencies were seen in 1 of 10 patients who had computed tomography of the head. Nerve conduction studies demonstrated abnormalities in 46% of the patients indicating that subclinical peripheral nerve dysfunction was common. Visual evoked responses were abnormal in only 1 patient but brainstem auditory evoked response studies showed some abnormality in 42% of the patients. The finding of HTLV-I antibodies in a significant number, and the isolation of HTLV-I from the blood in 6 of our black patients with noncompressive myelopathy, represents a substantial clinical advance. Future studies should define more clearly the role of the virus in this disorder.

Isolation of a new human herpesvirus producing a lytic infection of helper (CD4) T-lymphocytes in peripheral blood lymphocyte cultures--another cause of acquired immunodeficiency?

A new human helper (CD4) T-lymphotropic herpesvirus (HTLHV) was first isolated in February 1985 from the cultured peripheral blood lymphocytes (PBL) of a patient with the acquired immunodeficiency syndrome, and subsequently from the PBL of 1 patient with hairy cell leukaemia and 2 patients with lymphoproliferative disease associated with human T-lymphotropic virus type I infection. The viruses could be serially subcultured in umbilical cord PBL cultures in which they infected helper (CD4) T-lymphocytes producing multinucleate giant cells with intranuclear inclusions followed by cell lysis. Electron microscopy of infected cultures revealed that the isolates were herpesviruses. Specific DNA probing showed that the 4 isolates were related to one another but were distinct from cytomegalovirus, Epstein-Barr virus, Herpesvirus hominis types 1 and 2, and varicella-zoster virus. HTLHV lyses the same target cell as human immunodeficiency virus in PBL cultures suggesting that it may have a similar potential to cause acquired immune deficiency. The development of an unequivocally diagnostic serological test is a priority, so that the epidemiology and pathogenesis of HTLHV infection can be studied.

Isolation of human T-lymphotropic virus type I (HTLV-I) from a black South African with Kaposi's sarcoma.

Serological evidence for HTLV-I infection in the South African population has now been confirmed by the isolation of the virus from the peripheral blood lymphocytes of an adult Tsonga male. The subject was an indigenous black man from the south-eastern Transvaal who had suffered from Kaposi's sarcoma for a decade and in whom serum antibodies against HTLV-I were demonstrated. T-lymphocyte cell lines were established from his peripheral blood lymphocytes and shown to be productively infected with HTLV-I as evidenced by: the characteristic cell morphology; the typical viral morphogenesis on ultra-thin section electron microscopy; the viral genome in DNA extracted from the cell lines; characteristic reverse transcriptase activity and viral specific proteins in the cell culture supernatant fluids. Spread of infection occurs through sexual intercourse, from mother to child, and by blood transfusion. Donated blood should be screened to contain the spread of HTLV-I infection.

Isolation frequency and growth properties of HIV-variants: multiple simultaneous variants in a patient demonstrated by molecular cloning.

The biological properties and efficiency of isolation of different HIV (LAV/HTLV III, ARV, and AAV) subtypes were evaluated by recovering and growing HIV on fresh peripheral human lymphocytes. Cultures for virus isolation were performed from more than 180 German AIDS, ARC, LAS, and virus-exposed asymptomatic patients. The virus isolation rate depended on the state of health of the patients being close to 80% in AIDS patients, 30-40% in ARC/LAS patients, and lower in asymptomatic HIV seropositive patients. The cytopathic effects of the HIV isolates obtained on lymphocyte-cell cultures ranged from no effect to marked syncytia formation and cytopathogenicity. Marked differences were also observed in the replication rate of the various isolates. These properties were stable in all in vitro passages of the viruses performed so far and allowed to tentatively define four subtypes of HIV. In the majority of AIDS cases with neurological symptoms well-growing strains were obtained from peripheral blood, while all but two isolates from the cerebrospinal fluid of the same patients grew remarkably slowly and to only low titres on lymphocytes, suggesting that selection of variants for growth at specific sites of the body occurs. For one of the most cytopathogenic strains the influence of several variables of culture conditions (cell type, corticosteroids, IL-2, and polybrene) on virus replication was studied. Apart from polybrene, all parameters strongly influenced replication.

Immunological studies in haemophilic children.

A majority of haemophiliacs who have received large-pool plasma products within the past 5 years have been exposed to the putative agent of the acquired immunodeficiency syndrome (AIDS)--HIV. It is not known what the risk of infection is among patients in South Africa. A study was made of 39 children with congenital coagulation disorders attending the Red Cross War Memorial Children's Hospital Haemophilia Clinic. All but 3 had been treated exclusively with small-pool lyophilised cryoprecipitate or a factor IX concentrate prepared by local blood transfusion services. Three patients had also received imported non-heat-treated commercial products FEIBA (Immuno), Autoplex, Proplex (Hyland) or Factorate (Armour). Absolute lymphocyte counts were normal in all patients but the OKT4/OKT8 ratio was reduced below 1.0 in 9 children including 2 of the 3 who had received commercial plasma concentrates. A high titre of HIV antibody was present in 2 of the 38 patients tested. Both of these children had received imported plasma concentrates and 1 shows some features of the AIDS-related complex. These results suggest that haemophiliacs who receive non-heat-treated commercial concentrates may be at greater risk of HIV infection than patients treated with locally produced plasma products.

Mumps meningo-encephalitis.

Between July 1981 and June 1985, 49 cases (36 boys (73%) and 13 girls (27%] of mumps meningo-encephalitis confirmed by culture of the virus from the cerebrospinal fluid (CSF) were seen. Patients presented particularly in the late spring and early summer. A CSF cell count greater than 500 X 10(6)/l was obtained in 14 cases (28%), a total CSF protein greater than 0.8 g/l in 6 cases (12%) and a CSF glucose of less than 2.2 mmol/l in 2 cases (4%). Two cases are reported to illustrate the diagnostic problems which the infection may cause, particularly when the CSF changes resemble those of tuberculous meningitis. In 1 case neurogenic pulmonary oedema developed after a convulsion; this caused further diagnostic uncertainty.

Paediatric meningitis in the western Cape. A 3-year hospital-based prospective survey.

Between July 1981 and June 1984 1223 cases of meningitis were seen in the Department of Paediatrics, Tygerberg Hospital. The commonest form in each population group was aseptic meningitis. Positive viral cultures were obtained from the CSF in 108 cases. The median age of white children with aseptic meningitis, 64 months, was significantly greater than that of coloured children, 45 months (P greater than 0.0001), and black children, 26 months (P greater than 0.014). The commonest cause of confirmed bacterial meningitis was Neisseria meningitidis (140 cases; 11.5%), which continues to affect mainly young coloured children (median age 16.9 months). Resistance to sulphonamides was found among 21% of 114 N. meningitidis isolates. Among white children Haemophilus influenzae was responsible for 9 of the 18 cases of confirmed bacterial meningitis. Tuberculosis was responsible for 62 cases of meningitis (5%) and was a commoner cause of meningitis than either H. influenzae (47 cases) or Streptococcus pneumoniae (34 cases). Thirty-four confirmed cases of bacterial meningitis were seen in children less than 1 month old. Klebsiella species were responsible for 8 cases (24%), Escherichia coli for 6 cases (12%), group B beta-haemolytic Streptococcus for 5 cases (15%) while 4 cases each were due to N. meningitidis and Strept. pneumoniae.

Isolation of variants of lymphocytopathic retroviruses from the peripheral blood and cerebrospinal fluid of patients with ARC or AIDS.

LAV/HTLV-III/AAV viruses were isolated from 20 German patients with ARC/AIDS in order to investigate strain variation. Virus was isolated from the peripheral blood and/or cerebrospinal fluid (CSF) in umbilical cord peripheral blood lymphocyte (PBL) cultures. Isolates were identified by their cytopathic effect (CPE), by reverse transcriptase assays on cell-free infected culture supernatant fluid (SNF), and one or more of the following: immunofluorescence assays on infected cells for viral antigen using HTLV-III reference sera, Western blot analysis of cell-free infected culture SNF, electron microscopy of infected cells, and Southern blot restriction analysis and specific HTLV-III probing of DNA extracted from infected cultured PBL. The isolates could be classified into three groups according to differences in growth rate and cytopathic effect: Most showed what was regarded as the typical CPE, while some either grew rapidly and induced a striking CPE and others grew slowly with minimal CPE. In one patient, virus producing typical CPE was isolated from the peripheral blood while the isolate from his filtered cell-free CSF produced atypical slow CPE, suggesting that antigenic variation may occur with persistent infection or that superinfection may occur. Southern blot DNA restriction analysis of the DNA of three selected isolates showed that two of the isolates were similar but that the restriction pattern of all three differed from patterns previously published. Our results supplement the accumulating evidence of genetic variation among LAV/HTLV-III strains. The extent of this variation needs to be evaluated for any effect on the sensitivity of diagnostic tests, on the strategy of vaccine development, on tissue tropism by altering the viral surface receptor-binding sites, and possibly on the development of specific chemotherapy.

The acquired immune deficiency syndrome and related complex. A report of 2 confirmed cases in Cape Town with comments on human T-cell lymphotropic virus type III infections.

We report a case of acquired immune deficiency syndrome (AIDS) and one of AIDS-related complex presenting in Cape Town. The first patient was probably infected in the USA. In turn he infected the second patient by regular homosexual contact. Human T-cell lymphotropic virus type III (HTLV-III) was cultured, we believe for the first time in Africa, from the peripheral blood lymphocytes and a lymph node of our patient with AIDS. HTLV-III infection and high-risk groups in South Africa are discussed in comparison with those in the USA. It is suggested that HTLV-III infection and AIDS will increasingly affect women. Prevention of the spread of HTLV-III infection and AIDS is discussed in relation to close medical surveillance and the protection of blood and blood products from contamination. Counselling of patients with AIDS and persons infected with HTLV-III, general health education, and the protection of health care staff are important in preventing spread but beyond the scope of this article.

Isolation of a lymphadenopathy-associated virus from a patient with the acquired immune deficiency syndrome.

A virus similar to the lymphadenopathy-associated virus or human T-lymphotropic virus type III, which has been described in association with the acquired immune deficiency syndrome (AIDS) by several laboratories elsewhere in the world, was isolated from a Cape Town patient with lymphadenopathy and acquired immune deficiency. This virus has the characteristic morphogenesis and ultrastructure and its genome encodes the virus-specific p24 protein. It is T-lymphotropic and produces the characteristic cytopathic effect. It can be serially propagated in a human lymphocyte line of the T4+ phenotype. This isolate is being used in diagnostic immunofluorescence assays for virus-specific antibodies.

Serum antibodies to human T-cell leukaemia virus type I in different ethnic groups and in non-human primates in South Africa.

The prevalence of humoral antibodies to human T-cell leukaemia virus type I (HTLV-I) was investigated in different ethnic groups and in non-human primates in South Africa. Serum antibody levels were determined by enzyme-linked immunosorbent assay (ELISA) using either disrupted whole HTLV-I or purified p24 core protein (p24 HTLV-I) as antigens. ELISA was complemented by direct radio-immunoprecipitation assays using either purified iodinated p24 HTLV-I or radiolabelled lysates of an HTLV-producing cell line as antigen followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis of the immunoprecipitates, and by immunofluorescence using the HTLV-I-producing cell line HUT-102 as antigen. Antibodies were demonstrated in 3,5% of Asians, 3,5% of blacks and 4,1% of coloureds, but not in whites, and also in 29% of vervet monkeys and 33% of baboons. We conclude that HTLV-I or closely related viruses cause widespread infection in non-human primates in South Africa and in a lower percentage of humans, including apparently healthy blood donors. We are currently isolating retroviruses from seropositive reactors and investigating the possible relevance to disease in South Africa.