Prevalence and genetic characterisation of HTLV-1 and 2 dual infections in patients with pulmonary tuberculosis in Central-West Brazil

Human T-cell lymphotropic virus (HTLV) may impact the clinical course of tuberculosis (TB). Both infections are highly endemic in Brazil. The aim of this study was to assess the prevalence of HTLV-1/2 in TB patients in Central-West Brazil and to perform a genetic characterisation of the respective isolates. Of the 402 patients, six (1.49%) were positive for anti-HTLV and five (1.24%; 95% confidence interval: 0.46-3.05) were infected with HTLV-1/2. Genetic characterisation demonstrated that the four HTLV-1 isolates belonged to the Transcontinental subgroup A of the Cosmopolitan subtype a and that the HTLV-2 isolate belonged to subtype a (HTLV-2a/c). The prevalence of HTLV infection observed in this study is higher than that observed in local blood donors and the HTLV-1 and 2 subtypes identified are consistent with those circulating in Brazil.     

The presence of HTLV-I proviral DNA in the central nervous system of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis

Human T-lymphotropic virus type 1 (HTLV-I) is a pathogenic retrovirus associated with a chronic progressive myelopathy, termed HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP), as well as adult T-cell leukemia (ATL). A chronic inflammatory process has been implicated in HAM/TSP by a pathological study, but the exact mechanism still remains unknown. To understand better the complex mechanism of disease induction by HTLV-I, I studied the spreading pattern of HTLV-I in both peripheral blood mononuclear cells (PBMNCs) and central nervous system (CNS) tissues in patients with HAM/TSP using a quantitative polymerase chain reaction (PCR) method. My results indicated the primary event to be the efficient replication of HTLV-I in vivo, whereas HTLV-I is likely to be present in the constituent cells of the CNS in addition to the infiltrating mononuclear cells.     

Evaluation of Strongyloides stercoralis infection in patients with HTLV-1

Introduction: Individuals infected with the human T-lymphotropic virus type 1 (HTLV-1) may present severe and disseminated forms of Strongyloides stercoralis infection with low therapeutic response.Objective: To investigate the S. stercoralis infection and the seroprevalence of IgG anti-S. stercoralis antibodies in individuals infected with HTLV-1 attending the Reference Center for HTLV-1 (CHTLV) in Salvador, Bahia, Brazil.Materials and methods: We conducted a cross-sectional study in 178 HTLV-1-infected individuals treated at the HTLV specialized center between January, 2014, and December, 2018. The parasitological diagnosis of S. stercoralis was performed using the Hoffman, Pons and Janer, agar plate culture, and Baermann-Morais methods. The IgG anti-S. stercoralis detection was performed using an in house enzyme-linked immunosorbent assay (ELISA). The HTLV-1 infection was diagnosed using a commercial ELISA and confirmed by Western blot.Results: The frequency of S. stercoralis infection was 3.4% (6/178). Individuals infected with S. stercoralis from rural areas (50.0%; 3/6) also showed S. stercoralis hyperinfection (>3,000 larvae/ gram of feces). The frequency of circulating anti-S. stercoralis IgG antibodies was 20.8% (37/178). Conclusions: HTLV-1-infected people living in precarious sanitary conditions are more prone to develop severe forms of S. stercoralis infection. Considering the high susceptibility and unfavorable outcome of the infection in these individuals, the serological diagnosis for S. stercoralis should be considered when providing treatment.     

Curcumin increased the expression of c-FLIP in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease is a chronic neuroinflammatory disease, which is associated with HTLV-1 infection. There is no effective and satisfactory treatment of HAM/TSP. It has been shown that curcumin exhibits modulatory effects on apoptosis and cytotoxicity-related molecules in HAM/TSP patients. In the present study, we examined the effect of curcumin on the gene expression of caspase-8, caspase-10, and anti-apoptotic protein c-FLIP, in HAM/TSP patients. Furthermore, we compared the expression of these molecules between HAM/TSP and asymptomatic carriers. Real-time PCR was performed to examine the mRNA expression of caspase-8, caspase-10, and c-FLIP in studied groups. The mRNA expression of caspase-8 and caspase-10 was similar before and after curcumin treatment in HAM/TSP patients (P > 0.05). The mRNA expression of c-FLIPL and c-FLIPs was higher after curcumin treatment compared with before treatment and significant differences were observed between the two groups (P = 0.004 and P = 0.044, respectively). The mRNA expression levels of caspase-8, caspase-10, c-FLIPL, and c-FLIPs were not statistically significant between HAM/TSP patients and asymptomatic carriers (P < 0.05). In conclusion, our results showed that curcumin increased the expression of c-FLIP in HAM/TSP patients which might suggest that, this molecule is involved in the apoptosis of HTLV-1-infected cells. Further studies with large sample size could be useful to clarify the role of this supplement in HAM/TSP patients.     

Sandfly species diversity in association with human activities in the Kani tribe settlements of the Western Ghats,Thiruvananthapuram, Kerala, India

Sandfly prevalence in the Kani tribe settlements of Western Ghats in India was investigated. A total of 1,279 sandflies comprising 17 species was obtained. Sandfly abundance showed a negative correlation (r = -0.97, p = 0.003) with increase in altitudinal ranges from 0-1,000 m. When sandfly samples were grouped according to landscape characteristics of the location, the estimated Shannon-Weiner index (H) and species richness index (S) were high and species evenness index (J) was low in settlements located at 0-300 m altitudinal range. On the contrary, the values of H and J were high, while S was low at 301-600 m altitudinal range. With further increase in altitude, species diversity, S and J were low. Though the relative abundance of sandflies decreased with increase in altitude, the influence of altitudinal variation could not be attributed to determine sandfly diversity, since the number of sampling units were not uniform at all the altitudinal gradients due to nonavailability of suitable resting shelters. Sandfly species showed great aggregation at 0-300 m altitude interval, where not only the number of settlements were maximum (n = 19), but also the environmental conditions favoured sandfly abundance due to the concentration of tribal settlements, human dwellings and his activities.     

Upregulated expression of Iba1 molecules in the central nervous system of mice in response to neurovirulent influenza A virus infection

The present study deals with the expression of Iba1 molecules, a novel EF-hand Ca2+-binding protein, in the brain after stereotaxic introduction of the neurovirulent WSN strain of influenza A virus into the olfactory bulb of C57BL/6 mice. The virus selectively targeted the paraventricular and anterior olfactory nuclei. Infected neurons appeared as early as at day 3 post infection and degenerated and vanished by day 12. The Iba1 molecule was normally expressed in resting microglia. The overexpression of the Iba1 in microglial cells was detected at day 3 post infection, culminating at day 7 with a morphological activation. Iba1-immunopositive macrophages outnumbered microglia in the paraventricular and anterior olfactory nuclei, where the infected neurons had degenerated. Macrophages totally disappeared by day 12, and the Iba1-expression in microglia was reduced to a normal level by day 35. Lack of perforin predisposed the mice to long-term virus infection of the brain, leading to the prolonged Iba1-overexpression. These results show that the Iba1 is upregulated in the mouse brain in response to influenza virus infection and may play significant roles in the regulation of some immunological and pathophysiological functions of microglia during virus infection.     

血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用

【目的】研究血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用。【方法】用3株H5N1病毒滴鼻感染BALB/c小鼠,研究小鼠肺、脑、血中的病毒在感染后不同时间点的复制动态及病理进展,通过免疫组化和免疫荧光染色显示病毒在脑部血管内皮细胞及血管周围神经组织的感染情况。【结果】小鼠感染后病毒迅速在肺中高效复制,随即形成病毒血症;感染后第6天病毒在肺中的滴度和在血液样本中的检出率达到峰值,此时小鼠脑部才开始检测到病毒;小鼠脑内血管内皮细胞、脑血管周围神经组织的神经元和神经胶质细胞中可检测到流感病毒NP蛋白。【结论】血液播散可能是高致病性H5N1禽流感病毒进入中枢神经系统的途径之一。     

Characterization of the Serpin, α1-Antichymotrypsin, in Normal Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) from 20 male patients with nonneurologic disease (age 64.5 +/- 2.8 SEM) was analyzed for the presence of the serpin alpha 1-antichymotrypsin (alpha 1-ACT). A chymotrypsin-specific chromogenic substrate (succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) was used to examine the CSF samples. All CSF samples showed inhibitory activity ranging from 45 to 80% inhibition. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the samples revealed the presence of a 68-kDa protein migrating identical to authentic human plasma alpha 1-ACT. Complex formation was performed with iodinated bovine chymotrypsin for several representative CSF samples having the highest chymotrypsin inhibitory activity. Comparison was made with complex formation performed with commercially available authentic human plasma alpha 1-ACT. These studies showed the formation of complexes at 37 degrees C, regardless of whether the sample was subsequently boiled or not. In the case of CSF, two complex bands, mass smaller than with plasma alpha 1-ACT, were formed at the lower temperature whereas a single higher Mr band was formed when the samples were boiled. To determine whether cleavage of the serpin occurred, these studies were repeated using human neutrophil cathepsin G as target protease. A complex of approximately 90 kDa was formed with human alpha 1-ACT under these same conditions. alpha 1-ACT has been detected in senile amyloid plaques in brains of Alzheimer's disease patients, the only plasma serine protease inhibitor localized to these structures. Another serpin, protease nexin I, is also found in these plaques, but this inhibitor does not circulate in plasma.(ABSTRACT TRUNCATED AT 250 WORDS)     

我国HIV-1B'/C重组流行株Tat蛋白的表达、纯化及功能分析

根据全国HIV分子流行病学研究发现,B'和C亚型HIV-1在我国发生了重组,并以优势株形式在我国广泛流行.为了探讨这种HIV-1B'/C重组毒株tat基因的变异与其表型之间的关系,利用pET表达系统在大肠杆菌中高效表达了三种不同基因变异类型的Tat蛋白,重组蛋白占菌体总蛋白的26%,Western blot显示较好的反应原性,并通过金属鏊合层析纯化了目的蛋白.荧光素酶活性检测表明:体外表达的Tat蛋白具有明显的生物学活性,可以反式激活HIV LTR引导的报告基因的表达;三种Tat蛋白在激活活性上的差异与流行现场检测的病毒载量的高低存在明显的对应关系,说明tat基因的变异可以引起病毒生物学特性的改变,进而影响病毒的流行特征.此结果为进一步研究我国HIV重组毒株的基因变异特征及变异规律奠定了基础.     

Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.     

An efficient system for site-directed mutagenesis to make various mutants of the env gene of human immunodeficiency virus type 1

We developed an efficient system of site-directed mutagenesis for the envelope (env) gene of human immunodeficiency virus type 1 (HIV-1). To make a template plasmid for mutagenesis, pS+B/MluI, two independent selection markers, i.e. a unique restriction site, MluI, and an in-frame termination codon, were introduced into the region encoding the V3 domain of the env gene of an HIV-1 strain, NL4-3, which had been cloned in the pUC118 plasmid. When the env gene of the pS+B/MluI plasmid was mutated successfully using mutagenic primers such as synthetic oligonucleotides or PCR-amplified DNA fragments longer than 1.5 kbp, the plasmids became resistant to digestion with MluI and competent env genes were formed by suppression of the in-frame termination. Various site-directed mutants of the env gene of HIV-1 were accurately constructed in a short time even in the absence of proper restriction sites by this system. The system of site-directed mutagenesis we reported here will be a useful method to analyze the functions of variable genes like the env gene of HIV-1 precisely and rapidly.     

The VD1/RPD2 neuronal system in the central nervous system of the pond snail Lymnaea stagnalis studied by in situ hybridization and immunocytochemistry

Summary VD₁ and RPD₂ are two giant neuropeptidergic neurons in the central nervous system (CNS) of the pond snail Lymnaea stagnalis. We wished to determine whether other central neurons in the CNS of L. stagnalis express the VD₁/RPD₂ gene. To this end, in situ hybridization with the cDNA probe of the VD₁/RPD₂ gene and immunocytochemistry with antisera specific to VD₁ and RPD₂ (the 1-antiserum, Mab4H5 and ALMA 6) and to R₁₅ (the 1 and 16-mer antisera) were performed on alternate tissue sections. A VD₁/RPD₂ neuronal system comprising three classes of neurons (A1–A3) was found. All neurons of the system express the gene. Division into classes is based on immunocytochemical characteristics. Class A1 neurons (VD₁ and RPD₂) immunoreact with the 1-antiserum, Mab4H5 and ALMA 6. Class A2 neurons (1–5 small and 1–5 medium sized neurons in the visceral and right parietal ganglion, and two clusters of small neurons and 5 medium-sized neurons in the cerebral ganglia) immunoreact with the 1-antiserum and Mab4H5, but not with ALMA 6. Class A3 neurons (3–4 medium-sized neurons and a cluster of 4–5 small neurons located in the pedal ganglion) immunoreact with the 1-antiserum only. All neurons of the system are immunonegative to the R₁₅ antisera. The observations suggest that the neurons of the VD₁/RPD₂ system produce different sets of neuropeptides. A group of approximately 15 neurons (class B), scattered in the ganglia, immunostained with one or more of the antisera, but did not react with the cDNA probe in in situ hybridization.     

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-γ and iNOS expression in the CNS compared to BALB/c mice. The CNS of C57BL/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite.     

HIV-1 Vpr Protein Enhances Proteasomal Degradation of MCM10 DNA Replication Factor through the Cul4-DDB1[VprBP] E3 Ubiquitin Ligase to Induce G2/M Cell Cycle Arrest

Human immunodeficiency virus type 1 Vpr is an accessory protein that induces G₂/M cell cycle arrest. It is well documented that interaction of Vpr with the Cul4-DDB1[VprBP] E3 ubiquitin ligase is essential for the induction of G₂/M arrest. In this study, we show that HIV-1 Vpr indirectly binds MCM10, a eukaryotic DNA replication factor, in a Vpr-binding protein (VprBP) (VprBP)-dependent manner. Binding of Vpr to MCM10 enhanced ubiquitination and proteasomal degradation of MCM10. G₂/M-defective mutants of Vpr were not able to deplete MCM10, and we show that Vpr-induced depletion of MCM10 is related to the ability of Vpr to induce G₂/M arrest. Our study demonstrates that MCM10 is the natural substrate of the Cul4-DDB1[VprBP] E3 ubiquitin ligase whose degradation is regulated by VprBP, but Vpr enhances the proteasomal degradation of MCM10 by interacting with VprBP.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.