RESUMO
Cases of pox-like lesions in horses and donkeys have been associated with poxviruses belonging to different genera of the family Poxviridae. These include the orthopoxviruses vaccinia virus (VACV), horsepoxvirus (HPXV) and cowpoxvirus (CPXV), as well as a potentially novel parapoxvirus and molluscum contagiosum virus (MOCV). However, with the exception of VACV, HPXV and CPXV, the genomic characterization of the causative agents remains largely elusive with only single short genome fragments available. Here we present the first full-length genome sequence of an equine molluscum contagiosum-like virus (EMCLV) directly determined from skin biopsies of a horse with generalized papular dermatitis. Histopathological analysis of the lesions revealed severe epidermal hyperplasia with numerous eosinophilic inclusion bodies within keratinocytes. Virions were detected in the lesions in embedded tissue by transmission electron microscopy. The genome sequence determined by next- and third-generation sequencing comprises 166 843 nt with inverted terminal repeats (ITRs) of 3473 nt. Overall, 20 of the predicted 159 ORFs have no equivalents in other poxviruses. Intriguingly, two of these ORFs were identified to encode homologues of mammalian proteins involved in immune signalling pathways, namely secreted and transmembrane protein 1 (SECTM1) and insulin growth factor-like family receptor 1 (IGFLR1), that were not described in any virus family so far. Phylogenetic analysis with all relevant representatives of the Poxviridae suggests that EMCLV should be nominated as a new species within the genus Molluscipoxvirus.
Assuntos
Genoma Viral , Doenças dos Cavalos/virologia , Molluscipoxvirus/genética , Molluscipoxvirus/fisiologia , Infecções por Poxviridae/veterinária , Dermatopatias Virais/veterinária , Proteínas Virais/genética , Animais , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Cavalos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Molluscipoxvirus/isolamento & purificação , Vírus do Molusco Contagioso/genética , Fases de Leitura Aberta , Filogenia , Infecções por Poxviridae/patologia , Infecções por Poxviridae/virologia , Pele/patologia , Pele/virologia , Dermatopatias Virais/patologia , Dermatopatias Virais/virologia , Transcrição Gênica , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/genética , Sequenciamento Completo do GenomaRESUMO
A collection of 40 Bacillus anthracis strains mostly isolated from soil in Bulgaria between 1960 and 1980 were investigated. All strains were proven to be B. anthracis by culture and amplification of a B. anthracis-specific chromosomal marker. PCR demonstrated that in nine strains both virulence plasmids (pX01+/pX02+) and in four strains only one plasmid (pX02+) were present, whereas the majority of strains (n = 27) lacked both plasmids (pX01-/pX02-). Multi-locus-variable number of tandem repeat-analysis (MLVA) using 15 markers differentiated three genotypes. Comparison with typing data of more than 1,000 different B. anthracis strains revealed that Bulgarian genotypes affiliated with the A1.a cluster and form their own unique cluster different from clusters containing strains isolated in geographical proximity, e.g., Turkey, Georgia, Hungary, Albania or Italy. In addition, a new allele of one marker (vrrC2) was identified. Canonical single nucleotide polymorphisms analysis allocated 31 Bulgarian strains into the A.Br.008/009 and nine strains into the A.Br.WNA group, which is the first description of B. anthracis strains of the A.Br.WNA group on the Eurasian continent.
Assuntos
Bacillus anthracis/classificação , Bacillus anthracis/genética , Repetições Minissatélites , Tipagem Molecular , Polimorfismo de Nucleotídeo Único , Microbiologia do Solo , Bacillus anthracis/isolamento & purificação , Bulgária , Análise por Conglomerados , Genótipo , Plasmídeos/análise , Reação em Cadeia da Polimerase , Fatores de Virulência/genéticaRESUMO
In 1998, it was claimed that an 80-year-old glass tube intentionally filled with Bacillus anthracis and embedded in a sugar lump as a WWI biological weapon still contained viable spores. Today, genome sequencing of three colonies isolated in 1998 and subjected to phylogenetic analysis surprisingly identified a well-known B. anthracis reference strain isolated in the United States in 1981, pointing to accidental laboratory contamination.IMPORTANCE Next-generation sequencing and subsequent phylogenetic analyses are useful and reliable tools for the classification of recent and historical samples. The reliability of sequences obtained and bioinformatic algorithms has increased in recent years, and research has uncovered the identity of a presumed bioweapon agent as a contaminant.
Assuntos
Bacillus anthracis/classificação , Bacillus anthracis/isolamento & purificação , Armas Biológicas , Bacillus anthracis/genética , Filogenia , Análise de Sequência de DNA , Reino Unido , Estados UnidosRESUMO
We report here the complete genome sequence (GenBank accession no. KX268728) of tick-borne encephalitis strain HB171/11, isolated from an Ixodes ricinus tick from a natural focus where human neurological disease is rare. The strain shows unique characteristics in neuroinvasiveness and neurovirulence.
RESUMO
Following the recent discovery of two Pandoravirus species in 2013, a previously described endocytobiont isolated from the inflamed eye of a patient with keratitis was subjected to whole-genome sequencing (WGS). Here, we present the complete genome sequence of a new Pandoravirus isolate.
RESUMO
The intrathecal synthesis of antibodies against recombinant VP1, the major structural protein of JC virus (JCV), was studied in 18 patients with progressive multifocal leukoencephalopathy (PML) and in 31 patients with various neurological disorders. Two methods were used, the calculation of an antibody specific index (ASI) on one hand and an antigen-driven immunoblotting for the detection of oligoclonal antibodies on the other. Most PML patients displayed an elevated (> 1.5) ASI (78%) and anti-VP1 oligoclonal antibodies restricted to the cerebrospinal fluid (55%). Only two other patients (one case each of multiple sclerosis and of neuroborreliosis) also showed an intrathecal synthesis of anti-VP1 oligoclonal antibodies, likely as a result of a 'polyspecific' reaction within the central nervous system.
Assuntos
Anticorpos Antivirais/líquido cefalorraquidiano , Vírus JC/imunologia , Leucoencefalopatia Multifocal Progressiva/imunologia , Proteínas Estruturais Virais/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Proteínas Recombinantes/imunologiaRESUMO
Polymerase chain reaction assays are a powerful tool for detecting the presence of infectious genomes in the cerebrospinal fluid. Positive results always mean a current or pending infection of the central nervous system. Subacute (>7 days) or chronic infections induce an intrathecal humoral immune response and the appearance of oligoclonal IgG antibodies directed against the causal infectious agent. This local synthesis may be observed even in cases of severe systemic immunodeficiency. The use of polymerase chain reactions in combination with the detection of a specific intrathecal immune response should represent the most reliable strategy for the diagnosis of viral and chronic infections of the central nervous system. The authors describe their experience, using this approach, in herpetic encephalitis, acute and recurrent herpetic meningitis, varicella zoster-induced neurological diseases, cytomegalovirus encephalitis, progressive multifocal leukoencephalitis and tuberculous meningitis.
Assuntos
Infecções do Sistema Nervoso Central/líquido cefalorraquidiano , Infecções do Sistema Nervoso Central/diagnóstico , Immunoblotting , Reação em Cadeia da Polimerase , Idoso , Idoso de 80 Anos ou mais , Infecções do Sistema Nervoso Central/imunologia , Citomegalovirus/genética , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/líquido cefalorraquidiano , Infecções por Citomegalovirus/diagnóstico , Primers do DNA , Diagnóstico Diferencial , Encefalite por Herpes Simples/líquido cefalorraquidiano , Encefalite por Herpes Simples/diagnóstico , Encefalite por Varicela Zoster/líquido cefalorraquidiano , Encefalite por Varicela Zoster/diagnóstico , Feminino , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/isolamento & purificação , Humanos , Immunoblotting/métodos , Imunoglobulina G/líquido cefalorraquidiano , Recém-Nascido , Vírus JC/genética , Vírus JC/isolamento & purificação , Leucoencefalopatia Multifocal Progressiva/líquido cefalorraquidiano , Leucoencefalopatia Multifocal Progressiva/diagnóstico , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Simplexvirus/genética , Simplexvirus/isolamento & purificação , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/diagnósticoRESUMO
We amplified the mRNA for cytokines in peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) cells from 18 multiple sclerosis (MS) patients and 21 other neurological patients, using the reverse transcription polymerase chain reaction (RT-PCR). Radioactive hybridization of the amplified DNA allowed quantitation of mRNA levels. Expression of tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma and interleukin (IL)-10 mRNA was elevated in CSF cells from MS patients. IFN-gamma and IL-10 mRNA levels were higher in MS patients than in other inflammatory neurological diseases. mRNA coding for transforming growth factor (TGF)-beta was detectable in the majority of cases, with higher expression in CSF cells of MS and other inflammatory neurological diseases than in noninflammatory controls, and higher expression in PBMC of MS patients than in all other cases. In many MS patients both proinflammatory and immunoregulatory cytokine messages were detected in the CSF compartment without correlation with the clinical activity of the disease. In contrast, mRNA for the costimulatory molecule B7.1 was only detected in the CSF cells of some MS patients, who showed clinical signs of acute relapse at the time of the spinal tap.
Assuntos
Citocinas/metabolismo , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla/metabolismo , Citocinas/sangue , Citocinas/líquido cefalorraquidiano , Humanos , Interferon gama/metabolismo , Interleucinas/metabolismo , Esclerose Múltipla/sangue , Esclerose Múltipla/líquido cefalorraquidiano , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estimulação Química , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In normal conditions, albumin and immunoglobulin (Ig)G in the cerebrospinal fluid (CSF) originate from the blood, and there is no antibody production within the central nervous system. Up to 20% of CSF proteins are intrathecally synthesized, but the major fraction is blood-derived. The CSF/serum albumin quotient (QAlb) is the best marker of the blood-CSF barrier function. The corresponding immunoglobulin quotients (QIGG, QIGA, QIGM) are not linearly related to QAlb and their correlations are defined by an hyperbolic equation. This equation is used to discriminate between a blood-derived and a locally produced fraction of immunoglobulins in case of an intrathecal humoral immune response. The detection of CSF-specific oligoclonal IgG is more sensitive than the quantitative comparison between QIGG and QAlb. A further step is the determination of antibody indices and the detection of specific oligoclonal antibodies by antigen-driven immunoblots. CSF analysis remains a cornerstone for the diagnosis of various neurological disorders, including multiple sclerosis and infectious diseases of the central nervous system.
Assuntos
Líquido Cefalorraquidiano/química , Líquido Cefalorraquidiano/metabolismo , Química Clínica/métodos , Imunoglobulinas/sangue , Imunoglobulinas/líquido cefalorraquidiano , Sistema Nervoso Central/metabolismo , Humanos , Immunoblotting , Imunoglobulina G/metabolismo , Modelos Estatísticos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Cerebrospinal fluid (CSF) and serum samples from eight patients with bacteriologically proven (6) or clinically suspected (2) tuberculous meningitis were tested for the presence of anti-mycobacterial IgG antibodies by an affinity-mediated immunoblot technique. This technique is based on agarose gel isoelectric focusing of paired CSF and serum samples diluted to the same IgG concentration, and transfer of the specific IgG antibodies onto mycobacterial antigen-loaded nitrocellulose sheets. An intrathecal synthesis of anti-mycobacterial oligoclonal IgG antibodies, often superimposed on diffuse polyclonal production was shown in all patients but not in patients with tension headache or other neurological disorders. Similar results were obtained when a purified mycobacterial antigen, A60, was used for coating the nitrocellulose sheets in place of a whole mycobacterial homogenate, indicating that A60 was a major immunogen. The number of anti-mycobacterial oligoclonal IgG bands increased with time, and persisted for years even in clinically cured patients. Some IgG bands had no detectable anti-mycobacterial activity, at least with the antigens preparations used in this study. The demonstration of such anti-mycobacterial IgG bands in the CSF could be a useful adjunct for the diagnosis of tuberculous meningitis, especially in the case of negative cultures.
Assuntos
Anticorpos Antibacterianos/líquido cefalorraquidiano , Antígenos de Bactérias , Proteínas de Choque Térmico , Mycobacterium tuberculosis/imunologia , Tuberculose Meníngea/imunologia , Adolescente , Adulto , Proteínas de Bactérias/imunologia , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Immunoblotting , Imunoglobulinas/líquido cefalorraquidiano , Lactente , Masculino , Pessoa de Meia-Idade , Bandas Oligoclonais , Tuberculose Meníngea/diagnósticoRESUMO
Tuberculous meningitis cases were analyzed by an immunoblotting test based on Mycobacterium bovis BCG antigen complex A60. Anti-A60 immunoglobulin G (IgG) in cerebrospinal fluid (CSF) allowed early diagnosis, and concentrations decreased after recovery. In primary meningitis forms, anti-A60 IgGs were intrathecally synthesized and specific oligoclonal IgGs were present in CSF. In meningeal complications of pulmonary tuberculosis, there were matching titers of anti-A60 IgG in blood and CSF (mirror pattern). Correlation between CSF-restricted patterns and CSF pleocytosis was shown.
Assuntos
Antígenos de Bactérias/sangue , Antígenos de Bactérias/líquido cefalorraquidiano , Mycobacterium bovis/imunologia , Tuberculose Meníngea/líquido cefalorraquidiano , Tuberculose Meníngea/imunologia , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Humanos , Immunoblotting , Lactente , Masculino , Pessoa de Meia-IdadeRESUMO
Quantitative and qualitative techniques for assessment of the intrathecal humoral immune response in human African trypanosomiasis were compared, and their diagnostic potential for detection of the meningo-encephalitic stage of the disease was evaluated. Total and trypanosome specific immunoglobulin G (IgG) and IgM intrathecal synthesis were studied in paired cerebrospinal fluid (CSF) and blood samples of 38 trypanosomiasis patients and in three controls using Reiber's formulae. The presence of CSF-specific oligoclonal IgG and of trypanosome-specific antibodies was determined using iso-electric focusing followed by immunoblotting and antigen-driven immunoblots. The intrathecal IgG fraction (16% positive) and oligoclonal IgG detection (24% positive) were insensitive for detection of an intrathecal humoral immune response. Trypanosome-specific IgG synthesis, reflected by the IgG antibody index (AI) (26% positive), was confirmed by the presence of oligoclonal specific IgG (47% positive), but the latter was more sensitive. Although the detection technique failed for oligoclonal IgM, the intrathecal IgM fraction (42% positive) and the IgM AI (32% positive) indicated that the meningo-encephalitic stage of the disease is characterized by a dominant intrathecal IgM response, which was higher than the IgG response. The highest combination of diagnostic sensitivity and specificity for the meningo-encephalitic stage of trypanosomiasis was observed for quantitative IgM determinations.