Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

Polymorphisms in CCR5 chemokine receptor gene in Japan.

Mutations in the human CC chemokine receptor 5 (CCR5) gene may alter the expression or function of the protein product, thereby altering chemokine binding/signalling or human immunodeficiency virus type 1 (HIV-1) infection of the cells that normally express CCR5 protein. We performed a systematic survey of natural sequence variations in an 8.1-kb region of the entire CCR5 gene as well as CCR2V64I in 50 Japanese subjects and evaluated the effects of those variations on CCR5 promoter activity. We also analysed CCR5 promoters and CCR2V64I in 80 more Japanese and 186 Thais. There was no 32-bp deletion observed in Caucasians, but two types of non-synonymous substitutions were found in CCR5 genes of Japanese. Our results showed several novel characteristics of the CCR2-CCR5 haplotype structure that were not reported from studies on Caucasians and African-Americans. Specifically, we were able to show that the G allele at position -2852 from the CCR5 open reading frame in Japanese and Thais is the representative of the CCR5 promoter haplotype that was reported to be associated with rapid progression to acquired immune deficiency syndrome (AIDS) in HIV-1-infected individuals. Furthermore, nearly all non-synonymous polymorphisms in Japanese CCR5 occurred in haplotypes with elevated promoter activity. We thus hypothesized that there was a certain selective pressure favouring low levels of CCR5 expression during human evolution.

A three-base-deletion polymorphism in the upstream non-coding region of human interleukin 7 (IL-7) gene could enhance levels of IL-7 expression.

Interleukin 7 (IL-7) is a key factor in the survival, development and proliferation of B and T lymphocytes. Elevation of plasma IL-7 has been reported in several lymphopenia cases such as HIV-1 patients. After patients started to receive antiretroviral drugs and their CD4(+) cell counts had recovered, IL-7 in plasma decreased to normal levels. There are considerable variations in the levels of plasma IL-7 as well as the rate of CD4(+) T-cell restoration. Although pre-treatment plasma IL-7 levels have been shown to be prognostic for the rate of post-treatment CD4(+) T-cell restoration, the mechanisms responsible for the variations in plasma IL-7 and rate of CD4(+) T-cell restoration are still completely unknown. In the study here, we searched for genetic polymorphisms that might affect levels of IL-7 gene expression. For this purpose, we used 1658-bp PCR-amplified fragments of the IL-7 gene containing 1470 bp of the upstream non-coding region obtained from 151 Japanese and 234 Thai subjects. We found two novel human genetic polymorphisms in the upstream non-coding region of the IL-7 gene. The luciferase reporter assay demonstrated that one of those polymorphisms could increase the gene expression of IL-7. We speculate that this polymorphism, a three base ATC deletion just upstream of an out-of-frame ATG codon in the upstream non-coding region of the IL-7 gene, reduces the efficiency of translation from the upstream, out-of-frame ATG, resulting in increased translation efficiency from the authentic ATG of IL-7. Although the frequency of this allele is very low, it would be interesting to analyse this polymorphism in HIV-1-infected individuals with different rates of immune reconstitution after treatment with a highly active antiretroviral therapy.

Renal lesions in leprosy: a retrospective study of 199 autopsies.

In the present work, 199 patients with leprosy who underwent autopsy between 1970 and 1986 were retrospectively studied to determine the prevalence, types, clinical characteristics, and etiologic factors of renal lesions (RLs) in leprosy. Patients were divided into two groups: 144 patients with RLs (RL+) and 55 patients without RLs (RL-). RLs observed in 72% of the autopsied patients were amyloidosis (AMY) in 61 patients (31%), glomerulonephritis (GN) in 29 patients (14%), nephrosclerosis (NPS) in 22 patients (11%), tubulointerstitial nephritis (TIN) in 18 patients (9%), granuloma in 2 patients (1%), and other lesions in 12 patients (6%). AMY occurred most frequently in patients with lepromatous leprosy (36%; nonlepromatous leprosy, 5%; P < 0.01), recurrent erythema nodosum leprosum (33%; P < 0.02), and trophic ulcers (27%; 0.05 < P < 0.10). Ninety-seven percent of AMY was found in patients with lepromatous leprosy, 88% showed recurrent trophic ulcers, and 76% presented with erythema nodosum leprosum. NPS was found in older patients with arterial hypertension, neoplastic diseases, infectious diseases, and vasculitis associated with GN. Most patients with AMY presented with proteinuria (95%) and renal failure (88%). The most frequent causes of death were renal failure in patients with AMY (57%), infectious diseases in patients with GN (41%) and TIN (45%), and cardiovascular diseases in patients with NPS (41%). No difference in survival rates was observed among RL- patients and those with AMY, GN, NPS, or TIN.

Novel polymorphisms in human macrophage inflammatory protein-1 alpha (MIP-1alpha) gene.

Human macrophage inflammatory protein-1 alpha (MIP-1alpha) is a chemotactic cytokine, which binds to macrophages, T cells, and B cells affecting their activation. We found novel polymorphisms at four sites within MIP-1alpha gene in Japanese population: C to T in exon 2; A to G in intron 2; C to G and A to G in exon 3. They occurred on the same allele. Although MIP-1alpha effectively suppresses the replication of HIV-1 in vitro, we observed no statistically significant difference in the allele frequency of this polymorphism between HIV-1-infected and uninfected individuals in Japanese population. Since an increased transcription level of MIP-1alpha has been reported to be associated with inflammatory diseases such as atopic dermatitis, we also investigated the frequency of these polymorphisms among patients with atopic dermatitis, HIV-1-infected individuals (with a normal IgE level), and healthy donors. A small increase in ratio of homozygotes to other genotypes was observed in patients with atopic dermatitis (P = 0.04).

Naturally occurring deletional mutation in the C-terminal cytoplasmic tail of CCR5 affects surface trafficking of CCR5.

CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(-) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221-222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(-) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4(+) T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(-) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.

Polymorphism in the interleukin-4 promoter affects acquisition of human immunodeficiency virus type 1 syncytium-inducing phenotype.

The emergence of syncytium-inducing (SI) variants of human immunodeficiency virus type 1 (HIV-1) in infected individuals is an indicator of poor prognosis and is often correlated with faster CD4(+) cell depletion and rapid disease progression. Interleukin-4 (IL-4) is a pleiotropic cytokine with various immune-modulating functions including induction of immunoglobulin E (IgE) production in B cells, down-regulation of CCR5 (a coreceptor for HIV-1 non-SI [NSI] strains), and up-regulation of CXCR4 (a coreceptor for HIV-1 SI variants). Here we show that homozygosity of a polymorphism in the IL-4 promoter region, IL-4 -589T, is correlated with increased rates of SI variant acquisition in HIV-1-infected individuals in Japan. This mutation was also shown to be associated with elevated serum IgE levels in HIV-1-infected individuals, especially in those at advanced stages of disease. In contrast, neither a triallele polymorphism in IL-10, another Th2 cytokine, nor a biallele polymorphism in the RANTES promoter affected acquisition of the SI phenotype. This finding suggested that IL-4-589T increases IL-4 production in the human body and thus accelerates the phenotypic switch of HIV-1 from NSI to SI and possibly disease progression of AIDS.

A new polymorphism in the promoter region of the human interleukin-16 (IL-16) gene.

Interleukin 16 (IL-16) is a chemotactic cytokine which binds to CD4 and affects T cell activation. Here we report a novel single nucleotide polymorphism, T to C, in the promoter region of the IL-16 gene in two distinct Asian populations, Japanese and Thai. This mutation occurs at an allele frequency of approximately 22% and 18%, respectively. Although IL-16 potently suppresses replication of human immunodeficiency virus type 1 (HIV-1), we observed no significant difference in the allele frequency of this polymorphism between HIV-1-infected and non-HIV-1-infected individuals in both Asian populations. Since differential IL-16 levels have been reported to be associated with inflammatory diseases such as systemic lupus erythematosus, atopic dermatitis and allergic asthma, it would be of interest to analyze the allele frequency of this mutation in patients with these autoimmune and allergic diseases.

Polymorphism in RANTES chemokine promoter affects HIV-1 disease progression.

RANTES (regulated on activation normal T cell expressed and secreted) is one of the natural ligands for the chemokine receptor CCR5 and potently suppresses in vitro replication of the R5 strains of HIV-1, which use CCR5 as a coreceptor. Previous studies showed that peripheral blood mononuclear cells or CD4(+) lymphocytes obtained from different individuals had wide variations in their ability to secrete RANTES. These findings prompted us to analyze the upstream noncoding region of the RANTES gene, which contains cis-acting elements involved in RANTES promoter activity, in 272 HIV-1-infected and 193 non-HIV-1-infected individuals in Japan. Our results showed that there were two polymorphic positions, one of which was associated with reduced CD4(+) lymphocyte depletion rates during untreated periods in HIV-1-infected individuals. This mutation, RANTES-28G, occurred at an allele frequency of approximately 17% in the non-HIV-1-infected Japanese population and exerted no influence on the incidence of HIV-1 infection. Functional analyses of RANTES promoter activity indicated that the RANTES-28G mutation increases transcription of the RANTES gene. Taken together, these data suggest that the RANTES-28G mutation increases RANTES expression in HIV-1-infected individuals and thus delays the progression of the HIV-1 disease.

Location-specific, unequal contribution of the N glycans in simian immunodeficiency virus gp120 to viral infectivity and removal of multiple glycans without disturbing infectivity.

One of the striking features of human immunodeficiency virus, simian immunodeficiency virus (SIV), and other lentiviruses is extensive N glycosylation of the envelope protein. To assess the requirement of each N glycan for viral infectivity, we individually silenced all 23 N glycosylation sites in the gp120 subunit of SIVmac239 envelope protein by mutagenizing the canonical Asn-Xaa-Thr/Ser N glycosylation motif in an infectious molecular clone, attempted to rescue viruses from the clones, and compared the replication capability of the rescued viruses in MT4 cells. The mutation resulted in either the recovery of a fully infectious virus (category I); recovery of a faster-replicating virus, compared with the parental virus (category II); or no virus recovery (category III). These categorically different sites were not distributed randomly but were clustered. The sites of category I were localized largely in the N-terminal half, whereas the sites of categories II and III were localized in the C-terminal region, including the CD4 binding site, and the central part, including the C loop, respectively. To learn how far SIV can tolerate the removal of glycans, multiplex mutagenesis was also attempted. When they were appreciably distant from one another in the primary sequence, up to five sites could be silenced in combination without disturbing infectivity. On the other hand, it was difficult to silence contiguous sites. Thus, it appeared that a certain degree of sugar chain density over the local region had to be preserved. We discuss the potential utility of these variously deglycosylated mutants for clarifying the role of N glycans in SIV replication in vivo, as well as in the host response, and for designing vaccines and the generation of glycoprotein crystals.

Anti-HIV-1 and chemotactic activities of human stromal cell-derived factor 1alpha (SDF-1alpha) and SDF-1beta are abolished by CD26/dipeptidyl peptidase IV-mediated cleavage.

CD26 is a leukocyte-activation antigen that is expressed on T lymphocytes and macrophages and possesses dipeptidyl peptidase IV (DPPIV) activity, whose natural substrates have not been identified yet. CXC chemokines, stromal cell-derived factor 1alpha (SDF-1alpha) and 1beta (SDF-1beta), sharing the receptor CXCR-4, are highly efficacious chemoattractants for resting lymphocytes and CD34(+) progenitor cells, and they efficiently block the CXCR-4-mediated entry into cells of T cell line tropic strains of HIV type 1 (HIV-1). Here we show that both the chemotactic and antiviral activities of these chemokines are abrogated by DPPIV-mediated specific removal of the N-terminal dipeptide, not only when the chemokines are produced in transformed mouse L cell line to express human CD26 but also when they were exposed to a human T cell line (H9) physiologically expressing CD26. Mutagenesis of SDF-1alpha confirmed the critical requirement of the N-terminal dipeptide for its chemotactic and antiviral activities. These data suggest that CD26-mediated cleavage of SDF-1alpha and SDF-1beta likely occurs in human bodies and promotes HIV-1 replication and disease progression. They may also explain why memory function of CD4(+) cells is preferentially lost in HIV-1 infection. Furthermore, CD26 would modulate various other biological processes in which SDF-1alpha and SDF-1beta are involved.

Importance of the N-glycan in the V3 loop of HIV-1 envelope protein for CXCR-4- but not CCR-5-dependent fusion.

The V3 region of HIV-1 envelope protein possesses a single N-linked sugar chain, which is conserved in most HIV-1 strains. We studied its role in the life cycle of HIV-1 strains with different co-receptor usage. Removal of the glycan appeared to cause a marked reduction of CXCR-4- but not CCR-5-dependent virus entry. A basic amino acid substitution at the 11th position of V3 markedly compensated for the removal of the N-glycan. These results indicate that the N-glycan plays an important role for CXCR-4-dependent virus entry and that this role is exerted in a particular context of the peptide backbone.