Genetic diversity among Zygophyllum (Zygophyllaceae) populations based on RAPD analysis.

Zygophyllum species are succulent plants that are drought resistant and/or salt tolerant, growing under severe, dry climatic conditions. Despite their importance and abundance in the Mediterranean and Middle East regions, there is little information concerning molecular variations among species of this genus. Genetic diversity was assessed, using RAPD primers, of 12 populations of Z. coccineum, Z. album and Z. aegyptium collected from various locations in Egypt and Saudi Arabia. Yong leaves were used for DNA extraction. Genetic distances were calculated using Nei's method. A dendrogram was constructed based on the similarity data matrix by unweighted pair group method using arithmetic averages cluster analysis. Analysis with RAPD markers revealed genetic variation between and within populations of Zygophyllum. Zygophyllum coccineum showed higher levels of genetic variation and more unique alleles than the other species.

Regional variation in CCR5-Delta32 gene distribution among women from the US HIV Epidemiology Research Study (HERS).

The CCR5-Delta32 genotype is known to influence HIV-1 transmission and disease. We genotyped 1301 US women of various races/ethnicities participating in the HIV Epidemiologic Research Study. None was homozygous for CCR5-Delta32. The distribution of heterozygotes was similar in HIV-1 infected and uninfected women. Thirty-seven (11.8%) white, 28 (3.7%) blacks/African Americans (AA), seven (3.3%) Hispanics/Latinas, and one (6.6%) other race/ethnicity were heterozygous. The frequency of heterozygotes differed among sites for all races combined (P = 0.001). More heterozygotes were found in AA women in Rhode Island (8.9%) than in the other sites (3.1%) (P = 0.02), while heterozygosity in white women was most common in Maryland (28.6%) (P = 0.025). These regional differences could be accounted for by racial admixture in AAs, but not in whites. Regional variations should be considered when studying host genetic factors and HIV-1 in US populations.

HIV-specific cytotoxic T lymphocytes, HLA-A11, and chemokine-related factors may act synergistically to determine HIV resistance in CCR5 delta32-negative female sex workers in Chiang Rai, northern Thailand.

Understanding how highly HIV-exposed individuals remain HIV uninfected may be useful for HIV vaccine design and development of new HIV prevention strategies. To elucidate mechanisms associated with resistance to HIV infection, immunologic and genetic factors were examined in 14 HIV-exposed but persistently seronegative (HEPS) female sex workers from Chiang Rai, northern Thailand and in ethnically matched, HIV-positive (n = 9) and HIV-negative women (n = 9). The HEPS women were identified in a study of commercial sex workers who had an HIV-1 incidence of 20.3 per 100 person-years. A high frequency of HLA-A11 was observed in HEPS women (86%) compared with northern Thai controls (56%). HIV-specific cytotoxic T lymphocyte (CTL) lytic responses were detected in cryopreserved peripheral blood mononuclear cells (PBMCs), using HLA-A-matched subtype E HIV-1 peptides in four of seven (57%) HEPS women, eight of eight HIV-positive women, and zero of nine HIV-negative unexposed controls (p = 0.019 HEPS women vs. HIV-negative controls). CTL lysis levels were low, but responses were detected to peptides from Nef, Pol, Gag, and Env. Nef responses predominated in HEPS women. Compared with controls, HEPS women tended to have higher frequencies of CCR5 promotor 59402GG and SDF-1 3'UTR 801A genotypes known to influence HIV transmission or course of disease. HEPS women also had higher levels of spontaneous RANTES production by PBMCs than other groups. Each of these factors could potentially contribute to HIV resistance. As most HEPS women had one or more of these factors, they may prevent HIV infection synergistically by blocking HIV cell entry, delaying its dissemination, or killing HIV-infected cells.

Susceptibility of the porcine endogenous retrovirus to reverse transcriptase and protease inhibitors.

Porcine xenografts may offer a solution to the shortage of human donor allografts. However, all pigs contain the porcine endogenous retrovirus (PERV), raising concerns regarding the transmission of PERV and the possible development of disease in xenotransplant recipients. We evaluated 11 antiretroviral drugs licensed for human immunodeficiency virus type 1 (HIV-1) therapy for their activities against PERV to assess their potential for clinical use. Fifty and 90% inhibitory concentrations (IC(50)s and IC(90)s, respectively) of five nucleoside reverse transcriptase inhibitors (RTIs) were determined enzymatically for PERV and for wild-type (WT) and RTI-resistant HIV-1 reference isolates. In a comparison of IC(50)s, the susceptibilities of PERV RT to lamivudine, stavudine, didanosine, zalcitabine, and zidovudine were reduced >20-fold, 26-fold, 6-fold, 4-fold, and 3-fold, respectively, compared to those of WT HIV-1. PERV was also resistant to nevirapine. Tissue culture-based, single-round infection assays using replication-competent virus confirmed the relative sensitivity of PERV to zidovudine and its resistance to all other RTIs. A Gag polyprotein-processing inhibition assay was developed and used to assess the activities of protease inhibitors against PERV. No inhibition of PERV protease was seen with saquinavir, ritonavir, indinavir, nelfinavir, or amprenavir at concentrations >200-fold the IC(50)s for WT HIV-1. Thus, following screening of many antiretroviral agents, our findings support only the potential clinical use of zidovudine.

Evidence of a role for the Q151L mutation and the viral background in development of multiple dideoxynucleoside-resistant human immunodeficiency virus type 1.

The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1(HXB2)). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1(HXB2). In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1(HXB2) partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1(151M), HIV-1(151L) RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1(151L) was found to be less fit than HIV-1(151M), which may explain the preferential selection of HIV-1(151M) observed in vivo. The demonstrated ability of HIV-1(151L/68G) to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1(151L) on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.

Partial resistance to infection by R5X4 primary HIV type 1 isolates in an exposed-uninfected individual homozygous for CCR5 32-base pair deletion.

It is known that certain individuals remain persistently seronegative despite repeated exposure to HIV-1. Studies have shown that some exposed uninfected (EU) individuals who are homozygous for a 32-bp deletion in the CCR5 gene are resistant to infection with non-syncytium-inducing (R5) viruses. In the present investigation, we provide evidence that a highly exposed-uninfected individual with the CCR5 32-bp deletion (EUdelta32-1) also has partial resistance to syncytium-inducing (R5X4) HIV-1 viruses, when compared with unexposed-uninfected individuals with (UUdelta32-1 and UUdelta32-2) and without (UU-1 and UU-2) the 32-bp deletion. The partial resistance of EU cells was due neither to altered coreceptor expression, nor to specific mutation or deletion in the coding region of chemokine coreceptors CXCR4 and CCR3. While SDF-1, the ligand for CXCR4, blocked entry of R5X4 viruses to a similar extent in EUdelta32 and UUdelta32, there was a differential production of soluble factors by EUdelta32. Both CD4+ and CD8+ cells from EUdelta32-1 produced soluble factors that efficiently suppressed infection by HIV-1 R5X4 viruses when compared with supernatant from UUdelta32. These data provide evidence that additional soluble factors are involved in resistance to infection with R5X4 viruses.

Predicted and observed alleles of Plasmodium falciparum merozoite surface protein-1 (MSP-1), a potential malaria vaccine antigen.

The 19-kDa antigenic domain of Plasmodium falciparum merozoite surface protein (MSP)-1 is a potential malaria vaccine candidate. Based on the amino acid substitution, four known alleles, E-TSR (PNG-MAD20 type), E-KNG (Uganda-PA type), Q-KNG (Wellcome type), and Q-TSR (Indo type) of this domain have been identified. Using single or double crossover recombinational events, we predicted the existence of additional alleles of this antigen. The presence of the predicted alleles was determined in parasite isolates from western Kenya, by undertaking a cross-sectional and a longitudinal study. Of the ten predicted alleles, we have revealed the presence of three new alleles: E-KSG-L (Kenya-1 type); E-KSR-L (Kenya-2 type); and E-KNG-F (Kenya-3 type). The results of this study suggest that it may be possible to predict the complexity of the genetic makeup of natural parasite populations.

Host genes and HIV: the role of the chemokine receptor gene CCR5 and its allele.

Since the late 1970s, 8.4 million people worldwide, including 1.7 million children, have died of AIDS, and an estimated 22 million people are infected with human immunodeficiency virus (HIV)(1). During 1995 and 1996, major clinical and laboratory discoveries regarding HIV pathogenesis provided new hope for the prevention and treatment of HIV infection. One major discovery was that members of the chemokine receptor family serve as cofactors for HIV entry into cells. We describe the role of allelic polymorphism in the gene coding for the CCR5 chemokine receptor with regard to susceptibility to and disease course of HIV infection. We also examine the effect of this discovery on medical and public health practices.

Phylogenetic relationship among the malaria parasites based on small subunit rRNA gene sequences: monophyletic nature of the human malaria parasite, Plasmodium falciparum.

We analyzed the small subunit ribosomal RNA (SSUrRNA) gene sequences from 13 malaria species parasitic to humans, chimpanzees/gorillas, Old World monkeys, rodents, birds, and lizards in order to reconstruct the phylogenetic relationships among the Plasmodium species. The SSUrRNA genes of Plasmodium vivax and P. ovale were sequenced by the dideoxy method in our laboratory; other sequences were retrived from GenBank. These sequences were aligned with the SSUrRNA gene sequence of outgroup species, Paramecium and Toxoplasma. After gaps and ambiguous regions were deleted, the aligned sequences were used for phylogenetic analysis by maximum likelihood and distance methods. The tree defines two major clades, the first with the bird and reptile parasites, the second with the rest of the species. The two bird parasites, P. gallinaceum and P. lophurae, do not closely cluster with human, chimpanzee/gorilla, Old World monkey, or rodent parasites, but cluster with the lizard parasites. P. vivax clusters with three Old World monkey parasites, P. cynomolgi, P. fragile, and P. knowlesi in decreasing order of closeness. P. ovale, while in a separate clade, is more closely related to P. vivax than to P. malarie or P. falciparum. P. malariae and P. berghei do not closely cluster with any of the other clades or with each other. Statistical analysis proves that the placement of P. falciparum in the bird malaria clade is less likely than in the mammalian malaria clade. Our analysis reveals that: (1) human malaria parasites have an evolutionary independent origin; (2) P. falciparum is most closely related to P. reichenowi and did not arise from lateral transfer of a bird parasite, as was previously suggested; and (3) the lizard malaria parasites are true members of the genus Plasmodium.

Natural immune response to the C-terminal 19-kilodalton domain of Plasmodium falciparum merozoite surface protein 1.

We have characterized the natural immune responses to the 19-kDa domain of merozoite surface protein 1 in individuals from an area of western Kenya in which malaria is holoendemic. We used the three known natural variant forms of the yeast-expressed recombinant 19-kDa fragment that are referred to as the E-KNG, Q-KNG, and E-TSR antigens. T-cell proliferative responses in individuals older than 15 years and the profile of immunoglobulin G (IgG) antibody isotypes in individuals from 2 to 74 years old were determined. Positive proliferative responses to the Q-KNG antigen were observed for 54% of the individuals, and 37 and 35% of the individuals responded to the E-KNG and E-TSR constructs, respectively. Considerable heterogeneity in the T-cell proliferative responses to these three variant antigens was observed in different individuals, suggesting that the 19-kDa antigen may contain variant-specific T epitopes. Among responses of the different isotypes of the IgG antibody, IgG1 and IgG3 isotype responses were predominant, and the prevalence and levels of the responses increased with age. We also found that a higher level of IgG1 antibody response correlated with lower parasite density among young age groups, suggesting that IgG1 antibody response may play a role in protection against malaria. However, there was no correlation between the IgG3 antibody level and protection. Furthermore, we observed that although the natural antibodies cross-reacted with all three variant 19-kDa antigens, IgG3 antibodies in 12 plasma samples recognized only the E-KNG and Q-KNG constructs and not the E-TSR antigen. This result suggests that the fine specificity of IgG3 antibodies differentiates among variant-specific natural B-cell determinants in the second epidermal growth factor domain (KNG and TSR) of the antigen.

Blood and sporozoite stage-specific small subunit ribosomal RNA-encoding genes of the human malaria parasite Plasmodium vivax.

Malaria parasites, unlike other eukaryotes, have developmentally controlled distinct small subunit ribosomal RNA (SSUrRNA)-encoding genes (SSUrDNA), sporozoite stage-specific C and blood stage-specific A genes. This report describes characterization of the C and A forms of SSUrDNA from the human malaria parasite Plasmodium vivax. We have aligned and compared these sequences with the reported SSUrDNA sequences of other human malaria parasites to identify the regions with potential for diagnostic probes. The comparison revealed the presence of seven conserved regions (> or = 90% similarity), four highly variable regions (< 60% similarity) and three semiconserved regions. The analysis also revealed that the A and C genes of P. vivax share more similarity with each other, as compared to the A and C genes of P. falciparum. Comparison of the SSUrDNA of human, monkey and rodent malaria parasites revealed that the A genes share more similarity with each other than the C genes share with each other.

Monoclonal antibodies to the circumsporozoite protein repeats of a Plasmodium vivax-like human malaria parasite and Plasmodium simiovale.

We have recently described a Plasmodium vivax-like human malaria parasite. The circumsporozoite protein of this parasite is identical to that of a simian malaria parasite, P. simiovale, but different from two known types of P. vivax. Here, we describe the production of two monoclonal antibodies, Pam 172 and Pam 135, specific for the circumsporozoite protein repeat sequence APGANQEGGAA of the P. vivax-like malaria parasite. These two monoclonal antibodies recognized air-dried sporozoites of P. simiovale but not other human, simian, or rodent malaria parasites tested.

A study of polymorphism in the circumsporozoite protein of human malaria parasites.

We have characterized the circumsporozoite (CS) gene sequences of Plasmodium malariae China-1 CDC, isolated recently from a person who was infected 50 years ago in China, and P. vivax Chesson, isolated 48 years ago from a patient who had returned from New Guinea. These protein sequences were compared with the CS protein sequences of recently isolated P. vivax and P. malariae parasites. In a similar manner, we compared the previously characterized CS protein gene of P. falciparum clone 7G8, derived from a Brazilian isolate collected in 1980, with the CS protein genes of recent P. falciparum field isolates. In the case of the P. malariae CS protein gene, with the exception of an additional copy of major (NAAG) and minor (NDAG) repeat sequences and the presence of one copy of NDEG sequence, the China-1 CDC P. malariae parasite is similar to the Uganda-1 CDC isolate of 1982. In the nonrepeat region, changes were noted in two amino acid residues, one of which is also seen in a closely related monkey malaria parasite, P. brasilianum. In the case of P. vivax CS proteins, the nonrepeat region of the protein in Chesson strain shares identity with nearly 71% of the CS clones characterized from field isolates. In the P. falciparum CS proteins, the 7G8 CS protein sequence is identical to 75% of the genes of recent field isolates in the Th1R-N1 region. In the Th2R and Th3R regions, 34% and 55% of the CS clones analyzed, respectively, had changes at two amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Global occurrence of Plasmodium vivax-like human malaria parasite.

A Plasmodium vivax-like human malaria parasite was recently identified from Madang, a holoendemic malarious region in Papua New Guinea. The complete nucleotide sequence of the circumsporozoite (CS) protein gene of this parasite is presented here. The CS protein of this parasite has an 11-mer repeat sequence and is different from the other known CS protein genes of human malaria parasites. However, it is identical to the CS protein gene of a monkey malaria parasite, Plasmodium simiovale. This P. vivax-like malaria parasite was found in Sepik, another malarious region of Papua New Guinea, and in Brazil, Indonesia, and Madagascar. No pure isolate of this parasite was identified. Specific oligonucleotide probes were used to determine relative proportion of the P. vivax-like parasite in P. vivax (type 1 and type 2) mixed field isolates. Compared with P. vivax or Plasmodium falciparum, the circumsporozoite protein of P. vivax-like parasites showed markedly less polymorphism.

Identification of Plasmodium vivax-like human malaria parasite.

There are four species of human malarial parasite and several monkey ones, and in evolutionary terms the human and non-human primate plasmodia may be related. The tools of molecular biology have lately pointed to the existence of two types of Plasmodium vivax. Using specific oligonucleotides we have identified a human malaria parasite resembling P vivax under the microscope but with circumsporozoite (CS) protein differing from those of P vivax types 1 and 2. The CS protein of this "P vivax-like" malaria parasite is identical to that of P simiovale, a monkey parasite resembling P ovale, a human one, morphologically. Polyclonal serum raised against a partial repeat sequence of the P vivax-like malaria parasite specifically reacted with P simiovale sporozoites but not with those of P vivax types 1 and 2, P ovale, or P simium. Sera collected from people living in malaria endemic regions of Papua New Guinea and Brazil contained antibodies that specifically reacted with the synthetic peptides representing the repeat sequences of CS protein of this P vivax-like parasite. A comparison of the CS protein gene sequences of P simiovale and the P vivax-like malaria parasites with those of other primate parasites, data on serological cross-reactivity, and 18S ribosomal DNA analyses suggest that the new human malaria parasite described here is distinct from P falciparum, P malariae, P vivax, and P ovale, the four known species of human parasite.

Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.

Use of glass beads and CF 11 cellulose for removal of leukocytes from malaria-infected human blood in field settings.

Passage of malaria-infected blood through a two-layered column composed of acid-washed glass beads and CF 11 cellulose removes white cells from parasitized blood. However, because use of glass beads and CF 11 cellulose requires filtration of infected blood separately through these two resins and the addition of ADP, the procedure is time-consuming and may be inappropriate for use in the field, especially when large numbers of blood samples are to be treated. Our modification of this process yields parasitized cells free of contaminating leukocytes, and because of its operational simplicity, large numbers of blood samples can be processed. Our procedure also compares well with those using expensive commercial Sepacell resins in its ability to separate leukocytes from whole blood. As a test of usefulness in molecular biologic investigations, the parasites obtained from the blood of malaria-infected patients using the modified procedure yield genomic DNA whose single copy gene, the circumsporozoite gene, efficiently amplifies by polymerase chain reaction.

Wide distribution of the variant form of the human malaria parasite Plasmodium vivax.

We have found polymorphism in the repetitive and nonrepetitive regions of the sporozoite vaccine antigen, the circumsporozoite (CS) protein, in Plasmodium vivax malaria parasites from two geographically distant malaria endemic regions of the world. Like the recently described variant repeat sequence of P. vivax from Thailand, the CS protein repeat sequence of the variant P. vivax parasites from Papua New Guinea and Brazil is ANGA(G/D)(N/D)QPG, which differs from the previously identified CS repeat sequence, GDRA(D/A)GQPA, of P. vivax parasites from South America, Central America, and North Korea. Comparison of the P. vivax CS protein outside the repeat region revealed restricted polymorphism in regions that have exhibited T-cell immune function and sequence heterogeneity in the CS protein of Plasmodium falciparum. Our results show that P. vivax malaria parasites with the variant CS repeat sequences are widespread in nature and that the polymorphism in the CS protein of P. vivax is also present in the nonrepeat region.