Natural killer activity in mice infected with rabies virus and submitted to P-acnes (Propionibacterium acnes) as immunomodulator

The natural killer (NK) activity and lethality were evaluated in swiss mice experimentally infected with street rabies virus and submitted to immunomodulation by P. acnes (formerly Corynebacterium parvum). The infected animals were sacrificed at different times and spleen non-adherent cells were obtained through ficoll-hypaque gradient and depletion of glass-adherent cells. Immunosuppression was observed in rabies virus infected mice correlated with lower NK activity in clinically ill animals. Higher NK activity and percentual of survival were observed in the group submitted to P. acnes. The increased survival correlated with higher NK activity induced by P. acnes suggests a protective role of this natural barrier against rabies virus infection in mice. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

Distribution of rabies virus in infected mice, vaccinated and submitted to P-acnes as immunomodulator

The lethality and distribution of rabies virus were evaluated in swiss mice experimentally infected with street rabies virus, vaccinated and submitted to immunomodulation by P. acnes (formerly Corynebacterium parvum). The animals were sacrificed at different times,when the different tissues were collected and submitted to fluorescent antibody test (FAT) and mouse inoculation test (MIT). The group submitted to vaccination and P. acnes treatment presented a percentage of survival superior to that observed in infected mice only treated with P. acnes. Control infected animals had the lowest survival rates. The distribution of rabies virus in spleen of infected mice, vaccinated and submitted to P. acnes was superior to that verified in infected mice not treated with P. acnes. The increased survival correlated with the distribution of rabies virus in lymphoid tissues, could be interpreted as the consequence of P. acnes activity on macrophages. The results suggest the role of macrophages against rabies virus infection in mice and the importance of vaccination in the post expositive treatment of rabies. (C) 2002 Published by Elsevier B.V. Ltd.

Wild rabies virus detection by plaque assay from naturally infected brains in different species

A simple, sensitive and specific plaque assay protocol for the detection of wild type rabies virus in different species is described using confluent monolayers of chicken embryo cells in 6-well plates. Plaques are produced after application of either agarose or Sephadex G-100 overlay onto cell monolayers and incubation for 96 h after virus infection at 37 degreesC. The parameters affecting plaque appearance include cell seeding concentration, overlay composition and time of incubation after infection. Optimal conditions are seeding at a concentration of 4 x 10(6) cell/cm(3), incubation at 37 degreesC in 5% CO2 atmosphere during 96 h, using either 1% agarose or 2% Sephadex G-100 overlays. The described plaque assay would be a new valuable too] in conducting various quantitative investigations, since the chicken embryo cells are susceptible to rabies virus infection from all species studied. (C) 2004 Elsevier B.V. All rights reserved.

Detecção do Southern bean mosaic virus no Paraná, e separação do Bean rugose mosaic virus em feijoeiro

Em lavouras de feijoeiro (Phaseolus vulgaris) da cultivar Carioca Comum, no município de Londrina, Estado do Paraná, foram encontradas plantas com sintomas de necrose da haste, mosaico clorótico leve e porte reduzido, semelhantes aos sintomas causados por infecção viral. Exames de microscopia eletrônica revelaram a presença de partículas isométricas. em testes de imunodifusão dupla em gel de ágar os extratos foliares de plantas infetadas reagiram positivamente com anti-soro específico para o Southern bean mosaic virus (SBMV). O vírus foi purificado e a massa molecular de sua proteína capsidial foi estimada em 30 kDa, valor esperado para proteínas do capsídeo de vírus do gênero Sobemovirus. A gama de hospedeiras do SBMV isolado no Paraná foi restrita ao feijoeiro e a algumas cultivares de soja (Glycine max). A separação de dois vírus isométricos comuns em infecções mistas no feijoeiro foi possível através da reação de imunidade ao SBMV apresentada por Crotalaria sp, Chenopodium quinoa e Mucuna deeringiana, e da reação de susceptibilidade dessas mesmas hospedeiras ao Bean rugose mosaic virus (BRMV).

Coat protein and RNAs of Cole latent virus are not present within chloroplasts of Chenopodium quinoa-infected cells

O vírus latente da couve (Cole latent virus, CoLV), gênero Carlavirus, foi estudado, por microscopia eletrônica de transmissão e técnicas bioquímicas, em relação à ultra-estrutura das células infetadas de Chenopodium quinoa, e de sua associação com os cloroplastos. O CoLV foi observado como partículas dispersas pelo citoplasma entremeadas com vesículas membranosas e ribossomos e/ou como densas massas de partículas. Estes partículas reagiram por imunomarcação com anti-soro policlonal para o CoLV. Morfologicamente, cloroplastos, mitocôndrias e núcleos mostraram-se inalterados e partículas virais não foram encontradas dentro dessas organelas. Entretanto, agregados de partículas virais foram freqüentemente vistos em associação com a membrana externa dos cloroplastos e ocasionalmente com peroxissomos. Cloroplastos foram purificados em gradiente de Percoll e as proteínas e os RNA foram extraídos e analisados, respectivamente, por Western blot e Northern blot. Proteína capsidial e RNA associados ao CoLV não foram detectados nessa organela. Os resultados aqui obtidos indicam que a associação CoLV/cloroplastos, observada nos estudos de microscopia eletrônica, é possivelmente um evento casual dentro da célula hospedeira e que o vírus não se multiplica dentro dessa organela.

Viral infections in workers in hospital and research laboratory settings: a comparative review of infection modes and respective biosafety aspects

Objectives: To compare modes and sources of infection and clinical and biosafety aspects of accidental viral infections in hospital workers and research laboratory staff reported in scientific articles. Methods: PubMed, Google Scholar, ISI Web of Knowledge, Scirus, and Scielo were searched (to December 2008) for reports of accidental viral infections, written in English, Portuguese, Spanish, or German; the authors' personal file of scientific articles and references from the articles retrieved in the initial search were also used. Systematic review was carried out with inclusion criteria of presence of accidental viral infection's cases information, and exclusion criteria of absence of information about the viral etiology, and at least probable mode of infection.Results: One hundred and forty-one scientific articles were obtained, 66 of which were included in the analysis. For arboviruses, 84% of the laboratory infections had aerosol as the source; for alphaviruses alone, aerosol exposure accounted for 94% of accidental infections. of laboratory arboviral infections, 15.7% were acquired percutaneously, whereas 41.6% of hospital infections were percutaneous. For airborne viruses, 81% of the infections occurred in laboratories, with hantavirus the leading causative agent. Aerosol inhalation was implicated in 96% of lymphocytic choriomeningitis virus infections, 99% of hantavirus infections, and 50% of coxsackievirus infections, but infective droplet inhalation was the leading mode of infection for severe acute respiratory syndrome coronavirus and the mucocutaneous mode of infection was involved in the case of infection with influenza B. For blood-borne viruses, 92% of infections occurred in hospitals and 93% of these had percutaneous mode of infection, while among laboratory infections 77% were due to infective aerosol inhalation. Among blood-borne virus infections there were six cases of particular note: three cases of acute hepatitis following hepatitis C virus infection with a short period of incubation, one laboratory case of human immunodeficiency virus infection through aerosol inhalation, one case of hepatitis following hepatitis G virus infection, and one case of fulminant hepatitis with hepatitis B virus infection following exposure of the worker's conjunctiva to hepatitis B virus e antigen-negative patient saliva. of the 12 infections with viruses with preferential mucocutaneous transmission, seven occurred percutaneously, aerosol was implicated as a possible source of infection in two cases, and one atypical infection with Macacine herpesvirus 1 with fatal encephalitis as the outcome occurred through a louse bite. One outbreak of norovirus infection among hospital staff had as its probable mode of infection the ingestion of inocula spread in the environment by fomites.Conclusions: The currently accepted and practiced risk analysis of accidental viral infections based on the conventional dynamics of infection of the etiological agents is insufficient to cope with accidental viral infections in laboratories and to a lesser extent in hospitals, where unconventional modes of infection are less frequently present but still have relevant clinical and potential epidemiological consequences. Unconventional modes of infection, atypical clinical development, or extremely severe cases are frequently present together with high viral loads and high virulence of the agents manipulated in laboratories. In hospitals by contrast, the only possible association of atypical cases is with the individual resistance of the worker. Current standard precaution practices are insufficient to prevent most of the unconventional infections in hospitals analyzed in this study; it is recommended that special attention be given to flaviviruses in these settings. (C) 2011 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

The deep mycoses in HIV infection

The deep mycoses are uncommon infections, usually acquired from the inhalation or ingestion of fungal spores, sometimes from the soil in areas of endemicity, such as in the Americas and south-east Asia, or from decaying vegetable matter. They are also seen in immunocompromised persons and, increasingly, in HIV-infected persons. Respiratory involvement is frequent, with granuloma formation, and mucocutaneous involvement may be seen. Oral lesions of the deep mycoses are typically chronic but non-specific, though nodular or ulcerative appearances are common. Person-to-person transmission is rare. In HIV disease, the most common orofacial involvement of deep mycoses has been in histoplasmosis, cryptococcosis, aspergillosis and zygomycosis. Diagnosis is usually confirmed by lesional biopsy although culture may also be valuable. Treatment is with amphotericin or an azole.

Kaposi's sarcoma-associated herpesvirus infection and Kaposi's sarcoma in Brazil

Kaposi's sarcoma (KS) became a critical health issue with the emergence of acquired immunodeficiency syndrome (AIDS) in the 1980s. Four clinical-epidemiological forms of KS have been described: classical KS, endemic KS,iatrogenic KS, and AIDS-associated KS. In 1994, Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus type 8 was identified by Chang and colleagues, and has been detected worldwide at frequencies ranging from 80 to 100%. The aim of the present study was to evaluate the frequency of KSHV infection in KS lesions from HIV-positive and HIV-negative patients in Brazil, as well as to review the current knowledge about KS transmission and detection. For these purposes, DNA from 51 cases of KS was assessed by PCR: 20 (39.2%) cases of classical KS, 29 (56.9%) of AIDS-associated KS and 2 (3.9%) of iatrogenic KS. Most patients were males (7.5:1, M/F), and mean age was 47.9 years (SD = ± 18.7 years). As expected, HIV-positive KS patients were younger than patients with classical KS. On the other hand, patients with AIDS-associated KS have early lesions (patch and plaque) compared to classical KS patients (predominantly nodular lesions). This is assumed to be the result of the early diagnose of KS in the HIV-positive setting. KSHV infection was detected by PCR in almost all cases (48/51; 94.1%), irrespectively of the clinical-epidemiological form of KS. These results show that KSHV is associated with all forms of KS in Brazilian patients, a fact that supports the role of this virus in KS pathogenesis. © 2006 Brazilian Journal of Medical and Biological Research.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

Abordagem initial do paciente com mielopatia aguda não compressiva

Acute myelopathy are characterized by spinal cord dysfunction, developing sensitive, motor and autonomic signs and symptons. Since they are relatively rare, there are some difficulties to early diagnosis and to the beginning of the treatment. So, literature was reviewed to describe the main aetiologies of acute non compressive myelopathy: 1) demyelinating diseases; 2) systemic disease; 3) parainfectious; 4) delayed radiation myelopathy; 5) vascular myelopathy; 6) idiopatic and 7) vitamin B12 deficiency. Besides, we suggest an algorithm to initial approach of these patients and further aethiologic investigation. © Copyright Morelra Jr. Editora.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Pesquisa de anticorpos anti-virus da febre aftosa em espécies de animais domésticos e silvestres susceptíveis e não vacinadas, no Pantanal de Mato Grosso do Sul

Pós-graduação em Medicina Veterinária - FCAV

Correlations of CTLA-4 gene polymorphisms and hepatitis C chronic infection

Background: Cytotoxic T lymphocyte-associated factor 4 (CTLA-4) functions as a negative regulator of T cell-mediated immune response. Molecular changes associated to CTLA-4 gene polymorphisms could reduce its ability to suppress and control lymphocyte proliferation. Aims: To evaluate the frequency of CTLA-4 gene polymorphisms in chronic hepatitis C virus (HCV) infected patients and correlate to clinical and histological findings. Methods: We evaluated 112 HCV-infected subjects prospectively selected and 183 healthy controls. Clinical and liver histological data were analysed. - 318C > T, A49G and CT60 CTLA-4 single-nucleotide polymorphisms (SNPs) were studied by PCR-RFLP and AT(n) polymorphism by DNA fragment analysis by capillary electrophoresis in automatic sequencer. Results: Eight AT repetitions in 3' UTR region were more frequent in HCV-infected subjects. We found a positive association of -318C and + 49G with HCV genotype 3 (P = 0.008, OR 9.13, P = 0.004, OR 2.49 respectively) and an inverse association of both alleles with HCV genotype 1 (P = 0.020, OR 0.19, P = 0.002, OR 0.38 respectively). Allele + 49G was also associated to aminotransferases quotients > 3 (qALT, P = 0.034, qAST, P = 0.041). Allele G of CT60 SNP was also associated with qAST > 3 (P = 0.012). Increased number of AT repetitions was positively associated to severe necroinflammatory activity scores in liver biopsies (P = 0.045, OR 4.62). Conclusion: CTLA-4 gene polymorphisms were associated to HCVinfection. Eight AT repetitions were more prevalent in HCV-infected subjects. - 318C and + 49G alleles were associated to genotypes 1 and 3 infections and increased number of AT repetitions in 3' UTR region favoured severe necroinflammatory activity scores in liver biopsies.

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.

Sorveglianza dell’infezione da virus dell’epatite E (HEV) in Italia: from farm to table

L'epatite E è una malattia umana con caratteristiche di epatite acuta, causata da un ssRNA virus (HEV). Nel 1997, HEV è stato identificato per la prima volta nei suini (SwHEV). In seguito, diverse evidenze, tra cui la vicinanza genetica tra ceppi umani e suini, suggerirono la trasmissione zoonotica del virus. Nella presente tesi, l’identificazione di SwHEV è stata condotta mediante ricerca di porzioni di genoma virale attraverso RT-PCR. Dal 2011 al 2013, sono stati analizzati 343 campioni fecali (da 19 allevamenti) e 70 bili (da 2 macelli) prelevati da altrettanti suini, in diverse Regioni italiane. E’ stato inoltre condotto uno studio retrospettivo su 78 feci (da 3 allevamenti) raccolte nel 2000. Il virus è stato identificato nel 24,5% e 19,2% delle feci raccolte rispettivamente nel 2011-2013 e nel 2000. Nessuna bile è risultata positiva. Mediante sequenziamento del genoma intero di uno dei virus identificati, è stata condotta l’analisi filogenetica per valutarne il grado di correlazione con alti ceppi suini e umani. La presenza di HEV è stata valutata lungo la filiera di produzione suina, dal macello al punto vendita. Trentaquattro campioni di feci, fegato e muscolo sono stati raccolti in un macello da altrettanti suini sani (età:6-7 mesi). Quattordici feci e 2 fegati, sono risultati positivi per HEV. Sono state prelevate 129 salsicce sia allo stabilimento di trasformazione sia alla vendita, ma nessuna è risultata positiva. La presenza di HEV è stata valutata anche nelle salsicce di fegato, fresche e secche, acquistate presso una macelleria. Il genoma virale è stato rilevato nel 22,2% delle salsicce fresche e nel 4,3 % di quelle secche ma la vitalità del virus non è stata dimostrata. In conclusione, lo studio condotto ha confermato l’ampia circolazione di HEV nei suini e la possibile contaminazione dei prodotti carnei derivati, confermando la necessità di una continua sorveglianza.