Infection of the central nervous system with dengue virus 3 genotype I causing neurological manifestations in Brazil

Abstract: A case of dengue virus 3 (DENV-3) genotype I infection with neurological manifestations occurred in Belo Horizonte, Minas Gerais in October 2012. The serotype was detected by PCR, and the genotype was assessed by sequencing and phylogenetic analysis of the C-prM region. The virus causing neurological manifestations clustered with other sequences of DENV-3 genotype I. Because neurological manifestations of DENV are possibly misdiagnosed in Brazil, this study serves as an alert of the importance of DENV diagnoses in CNS infections.

RAP1 GTPase overexpression is associated with cervical intraepithelial neoplasia.

RAP1 (RAS proximate 1), a small GTP-binding protein of the RAS superfamily, is a putative oncogene that is highly expressed in several malignant cell lines and types of cancers, including some types of squamous cell carcinoma. However, the participation of RAP1 in cervical carcinogenesis is unknown. We conducted a cross-sectional study of paraffin-embedded cervical biopsies to determine the association of RAP1 with cervical intraepithelial neoplasia (CIN). Standard and quantitative immunohistochemistry assessment of RAP1 expression in fixed tissue was performed on 183 paraffin-embedded cervical biopsies that were classified as normal or non-dysplastic mucosa (NDM) (n = 33); CIN grade 1 (n = 84) and CIN grade 2/3 (n = 66). A gradual increase in RAP1 expression in NDM < CIN 1 < CIN 2/3 (p<0.001) specimens was observed and was in agreement with the histopathologic diagnosis. A progressive increase in the RAP1 expression levels increased the risk of CIN 1 [odds ratio (OR) = 3.50; 95% confidence interval (CI) 1.30-10.64] 3.5 fold and the risk of CIN 2/3 (OR = 19.86, 95% CI 6.40-70.79) nearly 20 fold when compared to NDM. In addition, stereotype ordinal regression analysis showed that this progressive increase in RAP1 expression more strongly impacted CIN 2/3 than CIN 1. Our findings suggest that RAP1 may be a useful biomarker for the diagnosis of CIN.

Caraparu virus induces damage and alterations in antioxidant defenses in the liver of BALB/c mice after subcutaneous infection.

Oxidative stress is a disturbance in the oxidant-antioxidant balance leading to potential cellular damage. Most cells can tolerate a mild degree of oxidative stress because they have a system that counteracts oxidation that includes antioxidant molecules such as glutathione (GSH) and superoxide dismutase (SOD). Disruption of the host antioxidant status has been recognized as an important contributor to the pathogenesis of many viruses. Caraparu virus (CARV) is a member of group C of the Bunyaviridae family of viruses. In South American countries, group C bunyaviruses are among the common agents of human febrile illness and have caused multiple notable outbreaks of human disease in recent decades; nevertheless, little is known about the pathogenic characteristics of these viruses. The purpose of this study was to examine the hepatic pathogenesis of CARV in mice and the involvement of oxidative stress and antioxidant defenses on this pathology. Following subcutaneous infection of BALB/c mice, CARV was detected in the liver, and histopathology revealed acute hepatitis. Increased serum levels of aspartate and alanine aminotransferases (AST/ALT) and greater hepatic expression of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) were found in infected animals. CARV infection did not alter the biomarkers of oxidative stress but caused an increase in GSH content and altered the expression and activity of SOD. This is the first report of an alteration of oxidative homeostasis upon CARV infection, which may, in part, explain the hepatic pathogenesis of this virus, as well as the pathogenesis of other Bunyaviridae members.

Differential upregulation of human 2'5'OAS genes on systemic sclerosis: Detection of increased basal levels of OASL and OAS2 genes through a qPCR based assay.

2'5'OAS are template-independent RNA polymerases with antiviral activity and important to homeostasis maintenance. Here we have developed quantitative PCR (qPCR) reactions for the detection of each individual 2'5'OAS human gene and used them to evaluate these gene levels in systemic sclerosis patients cells. The method was efficient for quantification of 2'5'OAS genes on human cells after interferon (IFN) treatment, and revealed that primary cells from patients with systemic sclerosis have increased basal levels of OASL and OAS2 genes. When treated, patients cells are able to induce all four 2'5'OAS genes. Our hypothesis is that abnormally circulating type I IFNs on the disease could be establishing a desensitized state on patients cells, making them refractory to subsequent IFN doses, and that OASL and OAS2 genes upregulation may be due to an IFN-independent stimulus. Further characterizing the biological activities of these genes, their induction pathways and their regulatory functions can lead to better understanding of systemic sclerosis molecular mechanisms and of their biological activities.

Study of Vaccinia and Cowpox viruses' replication in Rac1-N17 dominant-negative cells

Interfering with cellular signal transduction pathways is a common strategy used by many viruses to create a propitious intracellular environment for an efficient replication. Our group has been studying cellular signalling pathways activated by the orthopoxviruses Vaccinia (VACV) and Cowpox (CPXV) and their significance to viral replication. In the present study our aim was to investigate whether the GTPase Rac1 was an upstream signal that led to the activation of MEK/ERK1/2, JNK1/2 or Akt pathways upon VACV or CPXV' infections. Therefore, we generated stable murine fibroblasts exhibiting negative dominance to Rac1-N17 to evaluate viral growth and the phosphorylation status of ERK1/2, JNK1/2 and Akt. Our results demonstrated that VACV replication, but not CPXV, was affected in dominant-negative (DN) Rac1-N17 cell lines in which viral yield was reduced in about 10-fold. Viral late gene expression, but not early, was also reduced. Furthermore, our data showed that Akt phosphorylation was diminished upon VACV infection in DN Rac1-N17 cells, suggesting that Rac1 participates in the phosphoinositide-3 kinase pathway leading to the activation of Akt. In conclusion, our results indicate that while Rac1 indeed plays a role in VACV biology, perhaps another GTPase may be involved in CPXV replication.

Recombinant envelope protein-based enzyme immunoassay for IgG antibodies is comparable to neutralization tests for epidemiological studies of dengue infection.

Dengue virus (DENV) is the most prevalent arbovirus in the world, found mainly in tropical regions. As clinical manifestations present frequently as nonspecific febrile illness, laboratory diagnosis is essential to confirm DENV infections and for epidemiological studies. Recombinant envelope (E) antigens of four serotypes of DENV were used to develop an immunoglobulin G enzyme-linked immunosorbent assay (IgG-ELISA). To evaluate the IgG-ELISA, a panel of serum samples that had been tested previously by a plaque reduction neutralization test (PRNT) was investigated for the presence of anti-E antibodies against the four DENV serotypes. IgG-ELISA was found to have a sensitivity (91%) and specificity (98%) at a receiver-operating characteristic (ROC) optimized cutoff and demonstrated high performance as well as good indexes. A concordance of 97% was achieved between both assays, and only 21/704 (3%) samples were not concordant. The results of the present study demonstrate a moderate correlation between neutralizing antibody titers and IgG-ELISA values. These findings indicate that the recombinant protein-based IgG-ELISA is a suitable method for routine serodiagnosis, monitoring and seroepidemiological studies of DENV infections.

Virucidal activity of chemical biocides against mimivirus, a putative pneumonia agent.

BACKGROUND: Acanthamoeba polyphaga mimivirus (APMV), the largest known virus, has been studied as a putative pneumonia agent, especially in hospital environments. Despite the repercussions of the discovery of APMV, there has been no study related to the control of APMV and the susceptibility of this virus to disinfectants. OBJECTIVES: This work investigated the virucidal activity against mimivirus of chemical biocides commonly used in clinical practice for the disinfection of hospital equipment and rooms. STUDY DESIGN: APMV was dried on sterilized steel coupons, exposed to different concentrations of alcohols (ethanol, 1-propanol and 2-propanol) and commercial disinfectants (active chlorine, glutaraldehyde and benzalkonium chloride) and titrated in amoebas using the TCID50 value. The stability of APMV on an inanimate surface was also tested in the presence and absence of organic matter for 30 days. RESULTS: APMV showed a high level of resistance to chemical biocides, especially alcohols. Only active chlorine and glutaraldehyde were able to decrease the APMV titers to undetectable levels. Dried APMV showed long-lasting stability on an inanimate surface (30 days), even in the absence of organic matter. CONCLUSIONS: The data presented herein may help health and laboratory workers plan the best strategy to control this putative pneumonia agent from surfaces and devices.

Increased expression of 2'5'oligoadenylate synthetase and double-stranded RNA dependent protein kinase messenger RNAs on affected skin of systemic sclerosis patients.

Scleroderma or systemic sclerosis (SSc) is an autoimmune disorder of unknown aetiology characterized by excessive collagen synthesis and subsequent deposition on the skin and various internal organs. Interferons (IFNs) are well-known immunomodulators and inhibitors of collagen production. However, IFN therapy has been implicated in the development or exacerbation of several autoimmune diseases, including SSc. We analyzed the expression of several interferon-stimulated genes (ISGs) in affected skin of SSc patients (skin tissue and cultured skin fibroblasts). A set of ISGs (PKR, 2'5'OAS, MxA, and 6-16) was analyzed by real-time PCR using RNA extracted from cultured skin fibroblasts and skin tissue of normal individuals and SSc patients. Both normal and SSc affected skin cultured fibroblasts were sensitive to the IFN treatment and presented similar levels of all ISGs tested. However, PKR and 2'5'OAS mRNA expression levels were significantly higher in the affected skin tissue of SSc patients when compared to normal controls. These data suggest that the IFN system plays a role in the pathogenesis of SSc.

Relação entre a qualidade de amostras de escarro e o diagnóstico de micobacterioses por PCR / Relation between sputum samples quality and (PCR) for the diagnosis of micobacterioses

O objetivo do estudo foi relacionar a quantidade de células, a presença de leucócitos e outros elementos no escarro com a positividade da reação de PCR, para diagnóstico de micobacterioses. Trinta e nove amostras positivas por baciloscopia para bacilos álcool-ácido resistentes (BAAR), ou positivas em cultura em meio de Lõwenstein-Jensen foram analisadas pelo método de coloração de Gram, para avaliação da biota e quantificação de células e leucócitos. Foram também submetidas à reação de PCR, para identificação de micobactérias.As amostras, de acordo com os resultados do Gram, foram classificadas como: ótima, boa, aceitável, ruim ou muito ruim. Do total de amostras analisadas, 26 (66,7%) apresentaram PCR positiva concordando com a baciloscopia, 12 (30,7%) PCR negativa e baciloscopia ou cultura positiva e uma (2,6%) amostra não pôde ter seu resultado avaliado, pois o DNA apresentou-se degradado após sucessivas extrações. Dentre as amostras com PCR negativo e baciloscopia ou cultura positiva, 9 (23,0%) apresentaram qualidade muito ruim no Gram, 2 (5,2%) qualidade ruim e 1 (2,6%) apresentou qualidade aceitável. Os resultados mostraram que a qualidade do escarro influencia diretamente na positividade da PCR e que, quanto mais representativa do trato respiratório inferior for a amostra, melhor será o rendimento da detecção molecular...

Dominant culturable bacterial microbiota in the digestive tract of the American black vulture (Coragyps atratus Bechstein 1793) and search for antagonistic substances

As bactérias anaeróbias estritas e facultativas cultiváveis do trato digestivo de seis urubus (Coragyps atratus Bechstein 1793) foram isoladas e identificadas. Após a captura, as aves receberam uma alimentacão de baixa contaminacão durante uma semana para eliminar possíveis microorganismos alóctonos. A seguir, amostras colhidas na língua, estomago e intestinos foram pesadas, submetidas a diluicões decimais numa câmara anaeróbia, inoculadas em meios de cultura e incubadas em aerobiose e anaerobiose a 37ºC para enumeracão, isolamento e identificacão. As bactérias isoladas foram usadas posteriormente como produtoras e reveladoras para detectar possíveis fenômenos de antagonismo. A populacão bacteriana total ao longo do trato digestivo variou de 3,46 n 0,39 log UFC/g no estômago até 10,75 n 0,37 log UFC/g no intestino distal. Algumas bactérias foram isoladas pela primeira vez do trato digestivo de C. atratus: Actinomyces bovis, Lactobacillus cellobiosus, Micrococcus luteus, Neisseria sicca, Clostridium bifermentans, Enterobacter agglomerans, Peptostreptococcus sp., Sarcina sp., Serratia odorifera, and Shigella flexneri. Associacões entre microorganismos foram observadas durante o isolamento em dois casos, um envolvendo A. bovis e N. sicca, e o outro envolvendo A. bovis e um bastonete Gram-negativo. Hetero-, iso- e autoantagonismos foram observados, sugerindo um papel ecológico para esses microorganismos em termos de autocontrole populacional e de barreira ambiental no trato digestivo dessas aves.

Araçatuba virus: a vaccinialike virus associated with infection in humans and cattle.

We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Genetic variability of HIV-1 isolates from Minas Gerais, Brazil

We report results of nucleotide sequencing and phylogenetic analysis of the env gene of 11 HIV-1 isolates, in Belo Horizonte, Brazil. Ten isolates belonged to HIV-1 subtype B and one was a probable B/F mosaic. This putative B/F recombinant is similar but not identical in its nucleotide sequence to other B/F mosaics described in Brazil.

Preservaçäo de interferon de membrana amniótica humana / Preservation of the human amniotic membrane interferon

Foram realizadas investigaçöessobre a presença do interferon de membrana amniótica humana (IFN-AM) a fim de permitir pesquisas posteriores a respeito de sua purificaçäo, propriedades químicas e biológicas e ensaios clínicos.Testes de inativaçäo rápida foram realizados para ensejar resultados imediatos.IFN-AM foi preparado pela infecçäo de âmnios pelo vírus da doença de Newcastle.A atividade anti-vírica do IFN-AM foi determinada em um sistema de células Vero-vírus da encefalomiocardite de camundongo ou vírus Sindbis.Para os testes de inativaçäo, preparaçöes de IFN-AM contendo 0;1 e 2 por cento de soro de carneiro foram aquecidas em temperaturas de 35ºC a 100ºC em tempos variáveis e foi medida a atividade antivírica residual.Destes dados, foi calculada a constante de Arrhenius de queda de atividade pelo calor, que foi representada graficamente contra a temperatura absoluta.Os dados de temperaturas altas(45ºC e acima) foram extrapolados para temperaturas baixas(37ºC e abaixo).Os resultados mostraram que o IFN-AM preservou-se melhor em pH 2 em presença de soro.Os dados obtidos nas temperaturas de 45º, 55º e 65ºC permitiram o cálculo de constante de Arrhenius de queda para baixas temperaturas por extrapolaçäo.A -4, 2ºC näo deverá ocorrer perda de ativadade do IFN-AM.

A concentraçäo de interferon humano de membranas amnióticas: I- métodos fácicos / The concentration of human amniotic membrane interferon: I physical methods

A concentraçäo de preparaçöes de interferon humano de membranas amnióticas (IFN-MA) por métodos fásicos foi investigada. As preparaçöes foram obtidas a partir de âmnios infectados com o vírus da doença de Newcastle e o seu título de atividade anti-vírica foi determinado em células Vero, tendo como desafio o vírus da encefalomiocardite de camundongo ou o vírus Sindbis. A concentraçäo por diálise contra pressäo negativa, com as preparaçöes em pH2, mostrou recuperaçöes de IFN acima de 50//. O método, apesar de bastante lento, foi considerado conveniente para pequenos volumes (até 100ml). A diálise contra substância higroscápica (aquacide) na qual a água das preparaçöes retiradas por substância colocada externamente à membrana de diálise foi satisfatório, permitindo concentraçöes rápidas, de 6 a 10 vezes, com recuperaçöes acima de 50//, sobretudo se o material fosse dialisado posteriormente em água acidulada. A filtraçäo molecular utilizando membranas Millipore PSAC ou PTGC e Amicon näo concentradas previamente pela precipitaçäo com ácido tricloroáctico permitiu a concentraçäo sem perdas do IFN-MA

Purification of human anmiotic membrane interferon by affinity chromatography

A purificaçäo do interferon humano de membranas amnióticas foi realizada para se obter preparaçöes purificadas para sua caracterizaçäo físico-química, estudo de suas atividades biológicas e ensaios terapêuticos. O interferon foi produzido em doença de Newcastle ou o vírus Sendai. A determinaçäo da atividade anti-vírica foi feita em células Vero infectadas com o vírus da encefalomiocardite de camundongo. As preparaçöes de interferon foram concentradas por precipitaçäo por ácido tricloroactivo e cromatografadas em "Blue-Sepharose- concanavalina A, Sepharose ligada ao dipeptídeo triptofnio (trf) - triptofânio ao dipeptídeo triptofânio-tirosina (tyr). Com a "Blue-Sepharose", o pH ótimo de ligaçäo do interferon foi 5,4. A aplicaçäo em batelada mostrou que foram ligadas 37.000 unidades de interferon por ml de gel, sendo portanto, mais eficiente que a aplicaçäo em coluna, na qual somente foram ligadas 17.750 unidades por ml de gel. As fraçöes do pico de atividade foram purificadas 274 vezes, com 12//de recuperaçäo do interferon em um experimento, com um total de 78//de recuperaçäo e um fator de purificaçäo atingido foi de apenas 6,7; os ligantes Sepharose trp-trp e trp-tyr mostraram uma ligaçäo baixa do interferon, menor que 10.000 unidades por ml de gel

Produçäo de interferon humano de membranas amnióticas em microtécnica / Production of human amniotic interferon by microtechnique

Foi desenvolvido um sistema de produçäo de interferon humano de membranas amnióticas (IFN-MA) em microtécnica. Fragmentos de âmnio de 1,4 ou 2,2cm de diâmetro, em placas de microtécnica (24 câmaras), foram infectados por vírus Sendai e o IFN-MA resultante foi titulado. Maiores títulos (12.800 e 13.000 unidades por ml) foram obtidos quando utilizadas quantidades de meio de 0,5ml e 1,0ml); 6,4 unidades hemaglutinantes do vírus Sendai e um fragmento por câmara. Níveis de IFN-MA mais consistentes e altos foram alcançados na regiäo coriônica do âmnio (6.000 a 9.600 unidades/ml) quando comparados com aqueles da regiäo umbilical (860 a 2.500 unidades/ml) e reflexa (200 a 6.500 unidades/ml). O sistema com fragmentos de 2,2cm de diâmetro produziu, em média, quantidades de interferon/ml superiores (9.300 unidades/ml) quando comparado ao da produçäo do âmnio total (2.200 unidades/ml). Apesar da variabilidade nos títulos produzidos individualmente pelos fragmentos de 2,2cm de diâmetro, devido a econômia de tempo e de materiais e aos altos níveis de IFN resultantes, a microtécnica poderá se empregada quando um grande número de variáveis for investigada