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1.
J Virol ; 98(3): e0199523, 2024 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-38323813

RESUMEN

Historically, antibody reactivity to pathogens and vaccine antigens has been evaluated using serological measurements of antigen-specific antibodies. However, it is difficult to evaluate all antibodies that contribute to various functions in a single assay, such as the measurement of the neutralizing antibody titer. Bulk antibody repertoire analysis using next-generation sequencing is a comprehensive method for analyzing the overall antibody response; however, it is unreliable for estimating antigen-specific antibodies due to individual variation. To address this issue, we propose a method to subtract the background signal from the repertoire of data of interest. In this study, we analyzed changes in antibody diversity and inferred the heavy-chain complementarity-determining region 3 (CDRH3) sequences of antibody clones that were selected upon influenza virus infection in a mouse model using bulk repertoire analysis. A decrease in the diversity of the antibody repertoire was observed upon viral infection, along with an increase in neutralizing antibody titers. Using kernel density estimation of sequences in a high-dimensional sequence space with background signal subtraction, we identified several clusters of CDRH3 sequences induced upon influenza virus infection. Most of these repertoires were detected more frequently in infected mice than in uninfected control mice, suggesting that infection-specific antibody sequences can be extracted using this method. Such an accurate extraction of antigen- or infection-specific repertoire information will be a useful tool for vaccine evaluation in the future. IMPORTANCE: As specific interactions between antigens and cell-surface antibodies trigger the proliferation of B-cell clones, the frequency of each antibody sequence in the samples reflects the size of each clonal population. Nevertheless, it is extremely difficult to extract antigen-specific antibody sequences from the comprehensive bulk antibody sequences obtained from blood samples due to repertoire bias influenced by exposure to dietary antigens and other infectious agents. This issue can be addressed by subtracting the background noise from the post-immunization or post-infection repertoire data. In the present study, we propose a method to quantify repertoire data from comprehensive repertoire data. This method allowed subtraction of the background repertoire, resulting in more accurate extraction of expanded antibody repertoires upon influenza virus infection. This accurate extraction of antigen- or infection-specific repertoire information is a useful tool for vaccine evaluation.


Asunto(s)
Anticuerpos Antivirales , Infecciones por Orthomyxoviridae , Orthomyxoviridae , Animales , Ratones , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Linfocitos B/citología , Linfocitos B/inmunología , Células Clonales/citología , Células Clonales/inmunología , Regiones Determinantes de Complementariedad/inmunología , Vacunas contra la Influenza/inmunología , Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/sangre , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/virología
2.
PLoS Pathog ; 18(9): e1010770, 2022 09.
Artículo en Inglés | MEDLINE | ID: mdl-36107982

RESUMEN

Babesia are tick-borne protozoan parasites that can infect livestock, pets, wildlife animals, and humans. In the mammalian host, they invade and multiply within red blood cells (RBCs). To support their development as obligate intracellular parasites, Babesia export numerous proteins to modify the RBC during invasion and development. Such exported proteins are likely important for parasite survival and pathogenicity and thus represent candidate drug or vaccine targets. The availability of complete genome sequences and the establishment of transfection systems for several Babesia species have aided the identification and functional characterization of exported proteins. Here, we review exported Babesia proteins; discuss their functions in the context of immune evasion, cytoadhesion, and nutrient uptake; and highlight possible future topics for research and application in this field.


Asunto(s)
Babesia , Garrapatas , Animales , Animales Salvajes , Babesia/genética , Eritrocitos/parasitología , Humanos , Mamíferos , Análisis de Secuencia de ADN
3.
BMC Genomics ; 24(1): 483, 2023 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-37620766

RESUMEN

BACKGROUND: Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. RESULTS: Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named "ves1c". In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. CONCLUSIONS: We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.


Asunto(s)
Babesia , Animales , Caballos , Babesia/genética , Filogenia , Familia de Multigenes , Sistemas de Lectura Abierta , Cromosomas
4.
Arch Virol ; 168(2): 61, 2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36631547

RESUMEN

Although rabies is endemic in Malawi, there have been no studies in which rabies virus was systematically investigated and characterized in multiple animal hosts in that country. In order to provide molecular epidemiological data on rabies virus in Malawi, 683 suspected rabies case reports from 2008 to 2021 were examined, and 46 (dog = 40, cow = 5, and cat = 1) viable rabies-positive brain samples archived at the Central Veterinary Laboratory (CVL), Lilongwe, Malawi, were analyzed genetically. The results showed an increase in the submission of brain samples from 2008 to 2010, with the highest number of submissions observed in 2020. Of the 683 case reports analyzed for the period under review, 38.1% (260/683) (CI: 34.44 - 41.84) were confirmed by direct fluorescent antibody test. Among the confirmed cases, 65.4% (170/260) (CI: 59.23 - 71.09) were canine rabies. Further, phylogenetic analysis revealed that sequences from different animal hosts clustered together within the Africa 1b lineage, suggesting that the strains circulating in livestock are similar to those in domestic dogs. This finding supports the hypothesis that canine rabies is spilling over to livestock and emphasizes the need for further studies to provide data for effective control of rabies in Malawi.


Asunto(s)
Enfermedades de los Perros , Virus de la Rabia , Rabia , Femenino , Bovinos , Animales , Perros , Virus de la Rabia/genética , Rabia/epidemiología , Rabia/veterinaria , Filogenia , Malaui/epidemiología , Epidemiología Molecular , Enfermedades de los Perros/epidemiología , Ganado
5.
Emerg Infect Dis ; 28(4): 888-890, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35318934

RESUMEN

Leishmaniases are neglected tropical diseases of humans and animals. We detected Leishmania infantum in 3 mixed-breed dogs in Zambia that had no travel history outside the country. Our findings suggest presence of and probable emergence of leishmaniasis in Zambia, indicating the need for physicians and veterinarians to consider the disease during diagnosis.


Asunto(s)
Leishmania infantum , Leishmaniasis , Animales , Perros , Leishmaniasis/veterinaria , Enfermedades Desatendidas , Probabilidad , Zambia/epidemiología
6.
J Neuroinflammation ; 19(1): 13, 2022 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-35012562

RESUMEN

BACKGROUND: Postviral olfactory dysfunction (PVOD) following a viral upper respiratory tract infection (URI) is one of the most common causes of olfactory disorders, often lasting for over a year. To date, the molecular pathology of PVOD has not been elucidated. METHODS: A murine model of Toll-like receptor 3 (TLR3)-mediated upper respiratory tract inflammation was used to investigate the impact of URIs on the olfactory system. Inflammation was induced via the intranasal administration of polyinosinic-polycytidylic acid (poly(I:C), a TLR3 ligand) to the right nostril for 3 days. Peripheral olfactory sensory neurons (OSNs), immune cells in the olfactory mucosa, and glial cells in the olfactory bulb (OB) were analyzed histologically. Proinflammatory cytokines in the nasal tissue and OB were evaluated using the quantitative real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS: In the treated mice, OSNs were markedly reduced in the olfactory mucosa, and T cell and neutrophil infiltration therein was observed 1 day after the end of poly(I:C) administration. Moreover, there was a considerable increase in microglial cells and slight increase in activated astrocytes in the OB. In addition, qPCR and ELISA revealed the elevated expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and interferon-gamma both in the OB and nasal tissue. CONCLUSIONS: Taken together, the decreased peripheral OSNs, OB microgliosis, and elevated proinflammatory cytokines suggest that immunological changes in the OB may be involved in the pathogenesis of PVOD.


Asunto(s)
Inflamación/metabolismo , Bulbo Olfatorio/metabolismo , Infecciones del Sistema Respiratorio/metabolismo , Receptor Toll-Like 3/metabolismo , Animales , Modelos Animales de Enfermedad , Inflamación/inducido químicamente , Ratones , Microglía/metabolismo , Mucosa Olfatoria/metabolismo , Poli I-C/farmacología
7.
PLoS Pathog ; 16(10): e1008917, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33017449

RESUMEN

Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.


Asunto(s)
Babesia bovis/patogenicidad , Babesiosis/parasitología , Células Endoteliales/parasitología , Eritrocitos/parasitología , Animales , Babesia bovis/genética , Bovinos , Enfermedades de los Bovinos/parasitología , Proteínas de la Membrana , Parásitos/patogenicidad , Proteómica/métodos , Factores de Virulencia/genética
8.
Emerg Infect Dis ; 27(8): 2237-2239, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34287134

RESUMEN

Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.


Asunto(s)
Infecciones por Rickettsia , Rickettsia felis , Rickettsia , Siphonaptera , Animales , Humanos , Rickettsia/genética , Infecciones por Rickettsia/diagnóstico , Infecciones por Rickettsia/epidemiología , Zambia/epidemiología
9.
MMWR Morb Mortal Wkly Rep ; 70(8): 280-282, 2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33630820

RESUMEN

The first laboratory-confirmed cases of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV-2, in Zambia were detected in March 2020 (1). Beginning in July, the number of confirmed cases began to increase rapidly, first peaking during July-August, and then declining in September and October (Figure). After 3 months of relatively low case counts, COVID-19 cases began rapidly rising throughout the country in mid-December. On December 18, 2020, South Africa published the genome of a SARS-CoV-2 variant strain with several mutations that affect the spike protein (2). The variant included a mutation (N501Y) associated with increased transmissibility.†,§ SARS-CoV-2 lineages with this mutation have rapidly expanded geographically.¶,** The variant strain (PANGO [Phylogenetic Assignment of Named Global Outbreak] lineage B.1.351††) was first detected in the Eastern Cape Province of South Africa from specimens collected in early August, spread within South Africa, and appears to have displaced the majority of other SARS-CoV-2 lineages circulating in that country (2). As of January 10, 2021, eight countries had reported cases with the B.1.351 variant. In Zambia, the average number of daily confirmed COVID-19 cases increased 16-fold, from 44 cases during December 1-10 to 700 during January 1-10, after detection of the B.1.351 variant in specimens collected during December 16-23. Zambia is a southern African country that shares substantial commerce and tourism linkages with South Africa, which might have contributed to the transmission of the B.1.351 variant between the two countries.


Asunto(s)
COVID-19/diagnóstico , COVID-19/virología , SARS-CoV-2/genética , Adulto , COVID-19/epidemiología , Prueba de Ácido Nucleico para COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , SARS-CoV-2/aislamiento & purificación , Zambia/epidemiología
10.
J Hum Genet ; 65(1): 41-48, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31723216

RESUMEN

Neochlamydia strain S13 is an amoebal symbiont of an Acanthamoeba sp. The symbiont confers resistance to Legionella pneumophila on its host; however, the molecular mechanism underlying this resistance is not completely understood. Genome analyses have been crucial for understanding the complicated host-symbiont relationship but segregating the host's genome DNA from the symbiont's DNA is often challenging. In this study, we successfully identified a bimodal genomic structure in Neochlamydia strain S13 using PacBio RS II supported by ultra-long reads derived from MinION. One mode consisted of circular sequences of 2,586,667 and 231,307 bp; the other was an integrated sequence of the two via long homologous regions. They encoded 2175 protein-coding regions, some of which were implied to be acquired via horizontal gene transfer. They were specifically conserved in the genus Neochlamydia and formed a cluster in the genome, presumably by multiplication through genome replication. Moreover, it was notable that the sequenced DNA was obtained without segregating the symbiont DNA from the host. This is an easy and versatile technique that facilitates the characterization of diverse hosts and symbionts in nature.


Asunto(s)
Genoma Bacteriano , Bacterias Gramnegativas/genética , Análisis de Secuencia de ADN/instrumentación , Acanthamoeba/microbiología , Genómica/métodos , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/aislamiento & purificación , Filogenia , Análisis de Secuencia de ADN/métodos
11.
Int J Mol Sci ; 21(20)2020 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-33053895

RESUMEN

Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.


Asunto(s)
Aedes/genética , Aedes/virología , Virus del Dengue/fisiología , Dengue/virología , Proteínas del Choque Térmico HSP20/genética , Proteínas de Insectos/genética , Mosquitos Vectores/genética , Mosquitos Vectores/virología , Animales , Biología Computacional/métodos , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Transcriptoma , Replicación Viral
12.
BMC Genomics ; 20(1): 705, 2019 Sep 11.
Artículo en Inglés | MEDLINE | ID: mdl-31506064

RESUMEN

BACKGROUND: Infection with Toxoplasma gondii is thought to damage the brain and be a risk factor for neurological and psychotic disorders. The immune response-participating chemokine system has recently been considered vital for brain cell signaling and neural functioning. Here, we investigated the effect of the deficiency of C-C chemokine receptor 5 (CCR5), which is previously reported to be associated with T. gondii infection, on gene expression in the brain during T. gondii infection and the relationship between CCR5 and the inflammatory response against T. gondii infection in the brain. RESULTS: We performed a genome-wide comprehensive analysis of brain cells from wild-type and CCR5-deficient mice. Mouse primary brain cells infected with T. gondii were subjected to RNA sequencing. The expression levels of some genes, especially in astrocytes and microglia, were altered by CCR5-deficiency during T. gondii infection, and the gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed an enhanced immune response in the brain cells. The expression levels of genes which were highly differentially expressed in vitro were also investigated in the mouse brains during the T. gondii infections. Among the genes tested, only Saa3 (serum amyloid A3) showed partly CCR5-dependent upregulation during the acute infection phase. However, analysis of the subacute phase showed that in addition to Saa3, Hmox1 may also contribute to the protection and/or pathology partly via the CCR5 pathway. CONCLUSIONS: Our results indicate that CCR5 is involved in T. gondii infection in the brain where it contributes to inflammatory responses and parasite elimination. We suggest that the inflammatory response by glial cells through CCR5 might be associated with neurological injury during T. gondii infection to some extent.


Asunto(s)
Encéfalo/citología , Encéfalo/parasitología , Perfilación de la Expresión Génica , Receptores CCR5/deficiencia , Toxoplasma/fisiología , Animales , Astrocitos/metabolismo , Astrocitos/parasitología , Encéfalo/metabolismo , Técnicas de Inactivación de Genes , Ratones , Microglía/metabolismo , Microglía/parasitología , Receptores CCR5/genética
13.
Emerg Infect Dis ; 25(2): 370-372, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30666947

RESUMEN

In 2017, an unprecedented increase in febrile illness was observed in Dhaka, Bangladesh. Real-time reverse transcription PCR confirmed that 603 (40.2%) of 1,500 cases were chikungunya fever. Phylogenetic analysis revealed circulation of the non-A226V East/Central/South African genotype of chikungunya virus in Bangladesh.


Asunto(s)
Fiebre Chikungunya/epidemiología , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Brotes de Enfermedades , Genotipo , Bangladesh/epidemiología , Fiebre Chikungunya/historia , Virus Chikungunya/clasificación , Historia del Siglo XXI , Humanos , Filogenia , ARN Viral , Proteínas Virales/genética
14.
Cell Microbiol ; 20(9): e12852, 2018 09.
Artículo en Inglés | MEDLINE | ID: mdl-29691989

RESUMEN

The intracellular pathogen Legionella pneumophila influences numerous eukaryotic cellular processes through the Dot/Icm-dependent translocation of more than 300 effector proteins into the host cell. Although many translocated effectors localise to the Legionella replicative vacuole, other effectors can affect remote intracellular sites. Following infection, a subset of effector proteins localises to the nucleus where they subvert host cell transcriptional responses to infection. Here, we identified Lpw27461 (Lpp2587), Lpg2519 as a new nuclear-localised effector that we have termed SnpL. Upon ectopic expression or during L. pneumophila infection, SnpL showed strong nuclear localisation by immunofluorescence microscopy but was excluded from nucleoli. Using immunoprecipitation and mass spectrometry, we determined the host-binding partner of SnpL as the eukaryotic transcription elongation factor, Suppressor of Ty5 (SUPT5H)/Spt5. SUPT5H is an evolutionarily conserved component of the DRB sensitivity-inducing factor complex that regulates RNA Polymerase II dependent mRNA processing and transcription elongation. Protein interaction studies showed that SnpL bound to the central Kyprides, Ouzounis, Woese motif region of SUPT5H. Ectopic expression of SnpL led to massive upregulation of host gene expression and macrophage cell death. The activity of SnpL further highlights the ability of L. pneumophila to control fundamental eukaryotic processes such as transcription that, in the case of SnpL, leads to global upregulation of host gene expression.


Asunto(s)
Interacciones Huésped-Patógeno , Legionella pneumophila/patogenicidad , Proteínas de Transporte de Membrana/metabolismo , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Virulencia/metabolismo , Animales , Muerte Celular , Línea Celular , Núcleo Celular/química , Humanos , Inmunoprecipitación , Macrófagos/microbiología , Macrófagos/fisiología , Espectrometría de Masas , Microscopía Fluorescente , Unión Proteica , Transporte de Proteínas
15.
J Clin Microbiol ; 56(11)2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30158190

RESUMEN

Bovine babesiosis is a serious threat to the cattle industry. We prepared blood DNA samples from 13 cattle with clinical babesiosis from the Badulla (n = 8), Jaffna (n = 3), and Kilinochchi (n = 2) districts in Sri Lanka. These DNA samples tested positive in PCR assays specific for Babesiabovis (n = 9), Babesia bigemina (n = 9), and Babesiaovata (n = 1). Twelve cattle were positive for B. bovis and/or B. bigemina One cow was negative for the tested Babesia species but was positive for Babesia on microscopic examination; the phylogenetic positions of 18S rRNA and cytochrome oxidase subunit III gene sequences suggested that the cow was infected with Babesia sp. Mymensingh, which was recently reported from a healthy cow in Bangladesh. We then developed a novel Babesia sp. Mymensingh-specific PCR assay and obtained positive results for one other sample. Analysis of gene sequences from the cow with positive B. ovata-specific PCR results demonstrated that the animal was infected not with B. ovata but with Babesia sp. Hue-1, which was recently reported from asymptomatic cattle in Vietnam. The virulence of Babesia sp. Hue-1 is unclear, as the cow was coinfected with B. bovis and B. bigemina However, Babesia sp. Mymensingh probably causes severe clinical babesiosis, as it was the sole Babesia species detected in a clinical case. The present study revealed the presence of two bovine Babesia species not previously reported in Sri Lanka, plus the first case of severe bovine babesiosis caused by a Babesia species other than B. bovis, B. bigemina, and Babesiadivergens.


Asunto(s)
Babesia/genética , Babesia/aislamiento & purificación , Babesiosis/microbiología , Enfermedades de los Bovinos/microbiología , Animales , Babesia/clasificación , Babesia/citología , Babesia bovis/genética , Babesia bovis/aislamiento & purificación , Babesiosis/epidemiología , Babesiosis/patología , Babesiosis/fisiopatología , Bovinos , Enfermedades de los Bovinos/patología , Enfermedades de los Bovinos/fisiopatología , ADN Protozoario/genética , Femenino , Filogenia , Reacción en Cadena de la Polimerasa/veterinaria , Proteínas Protozoarias/genética , ARN Ribosómico 18S/genética , Análisis de Secuencia de ADN/veterinaria , Sri Lanka/epidemiología
16.
Malar J ; 17(1): 217, 2018 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-29843734

RESUMEN

BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.


Asunto(s)
Malaria Falciparum/clasificación , Mutación , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Humanos , Indonesia , Malaria Falciparum/parasitología , Nanoporos , Plasmodium falciparum/aislamiento & purificación
17.
Exp Parasitol ; 185: 1-9, 2018 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-29307564

RESUMEN

Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain.


Asunto(s)
Genómica , Malaria/parasitología , Plasmodium berghei/patogenicidad , Proteómica , Animales , Cromatografía Liquida , ADN Protozoario/química , ADN Protozoario/genética , Eritrocitos/parasitología , Femenino , Malaria/mortalidad , Ratones , Ratones Endogámicos C57BL , Parasitemia/parasitología , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Proteínas Protozoarias/genética , Transcripción Reversa , Eliminación de Secuencia , Organismos Libres de Patógenos Específicos , Espectrometría de Masas en Tándem , Virulencia
18.
BMC Genomics ; 18(1): 832, 2017 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-29078748

RESUMEN

BACKGROUND: Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans. RESULTS: The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia. CONCLUSIONS: Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites.


Asunto(s)
Babesia/clasificación , Babesia/genética , Evolución Molecular , Genoma de Protozoos , Genómica , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Genómica/métodos , Anotación de Secuencia Molecular , Transcriptoma , Secuenciación Completa del Genoma
19.
Genome Res ; 24(9): 1433-44, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25091627

RESUMEN

To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.


Asunto(s)
Genoma Humano , Genoma de Protozoos , Malaria/genética , Plasmodium falciparum/genética , Transcriptoma , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adolescente , Adulto , Antimaláricos/uso terapéutico , Estudios de Casos y Controles , Niño , Preescolar , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Resistencia a Medicamentos/genética , Etiquetas de Secuencia Expresada , Femenino , Interacciones Huésped-Parásitos/genética , Humanos , Inmunidad Innata/genética , Lactante , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Malaria/diagnóstico , Malaria/tratamiento farmacológico , Masculino , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Plasmodium falciparum/patogenicidad , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-jun/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Virulencia/genética
20.
BMC Infect Dis ; 17(1): 621, 2017 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-28903726

RESUMEN

BACKGROUND: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. METHODS: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. RESULTS: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. CONCLUSIONS: Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.


Asunto(s)
Malaria/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Cartilla de ADN , Humanos , Indonesia , Límite de Detección , Malaria Falciparum/diagnóstico , Malaria Vivax/diagnóstico , Técnicas de Diagnóstico Molecular/instrumentación , Técnicas de Diagnóstico Molecular/métodos , Nanoporos , Técnicas de Amplificación de Ácido Nucleico/instrumentación , Plasmodium/genética , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S
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