Magnetic purification of biotinylated cDNA removes false priming and ensures strand-specificity of RT-PCR for enteroviral RNAs

The detection of replicative intermediate RNAs as markers of active replication of RNA viruses is an essential tool to investigate pathogenesis in acute viral infections, as well as in their long-term sequelae. In this regard, strand-specific PCR has been used widely to distinguish (-) and (+) enteroviral RNAs in pathogenesis studies of diseases such as dilated cardiomyopathy. It has been generally assumed that oligonucleotide-primed reverse transcription of a given RNA generates only the corresponding specific cDNA, thus assuring the specificity of a PCR product amplified from it. Nevertheless, such assumed strand-specificity is a fallacy, because falsely primed cDNAs can be produced by RNA reverse transcription in the absence of exogenously added primers, (cDNA(primer)(-)), and such falsely primed cDNAs are amplifiable by PCR in the same way as the correctly primed cDNAs. Using as a prototype the coxsackievirus B5 (CVB5), a (+) strand RNA virus, it was shown that cDNA(primer)(-) renders the differential detection of viral (-) and (+) RNAs by conventional PCR virtually impossible, due to gross non-specificity. Using in vitro transcribed CVB5 RNAs (+) and (-), it was shown that cDNA(primer)(-) could be removed effectively by magnetic physical separation of correctly primed biotinylated cDNA. Such strategy enabled truly strand-specific detection of RNA (-) and (+), not only for CVB5, but also for other non-polio enteroviruses. These findings indicate that previous conclusions supporting a role for the persistence of actively replicating enterovirus in the pathogenesis of chronic myocarditis should be regarded with strong skepticism and purification of correctly primed cDNA should be used for strand-specific PCR of viral RNA in order to obtain reliable information on this important subject. (C) 2009 Elsevier B.V. All rights reserved.

Hemorrhagic Cystitis Secondary to Adenovirus or Herpes Simplex Virus Infection Following Renal Transplantation: Four Case Reports

Viral infections are common complications following renal transplantation. However, there have been few reported cases of viral cystitis secondary to herpes simplex virus or adenovirus infection. Herein, we have reported four cases of hemorrhagic cystitis secondary to infections with herpes simplex virus and adenovirus following renal transplantation. The etiology was adenovirus in three cases and herpes simplex virus in the remaining case. In all four cases, the primary cause of the renal dysfunction was diabetic nephropathy. All four patients presented with a clinical profile characterized by dysuria, pollakiuria, macroscopic hematuria, and graft dysfunction. Three of the four patients developed these symptoms within the first 3 months after renal transplantation. In all four cases, there was an increase, albeit slight, in creatinine levels, which returned to normal or near-normal values upon resolution of the symptoms. Acute cellular rejection was observed in only one case. Although rare, hemorrhagic cystitis secondary to infection, which typically occurs early in the posttransplant period, causes pronounced symptoms. The infection appears to be self-limiting, resolving completely within 4 weeks.

Inhibition of Expression of HTLV-1 Structural Genes Mediated by Short Hairpin RNA In Vitro

Background: Human T-lymphotropic virus 1 (HTLV-1) is associated with the T-cell malignancy known as adult T-cell leukemia! lymphoma (ATLL) and with a disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, the treatment of these diseases is based on symptom relief. RNA interference (RNAi) technology has been described as an efficient mechanism for development of new therapeutic methods. Thus, the aim of this study was to evaluate the inhibition of HTLV-1 structural proteins using short hairpin RNAs (shRNAs) expressed by non-viral vectors. Materials and Methods: Reporter plasmids that express enhanced green fluorescent protein-Gag (EGFP-Gag) and EGFP-Env fusion proteins and vectors that express shRNAs corresponding to the HTLV-1 gag and env genes were constructed. shRNA vectors and reporter plasmids were simultaneously transfected into HEK 293 cells. Results: Fluorescence microscopy, flow cytometry and real-time PCR showed that shRNAs were effective in inhibiting the fusion proteins. Conclusion: These shRNAs are effective against the expression of structural genes and may provide an approach to the development of new therapeutic agents.

Respiratory Infection, Exposure to Mouse Allergen and Breastfeeding: Role in Recurrent Wheezing in Early Life

Background: Environmental factors may influence the development of allergen sensitization and asthma. The aim of this study was to evaluate the role of endotoxin and allergen exposure in early life as a risk factor for recurrent wheezing. Methods: One hundred and four infants from low-income families, at high risk of asthma, were enrolled at birth. Dust samples were collected from the bedding and bedroom floor within 6 months after birth. Recurrent wheezing was defined as 3 or more wheezing episodes in the past year. Endotoxin was determined by Limulus amebocyte lysate assay, and major indoor allergens were quantitated by ELISA in dust extracts. IgE antibodies were measured by ImmunoCAP at 30 months of age. Results: At 30 months, 51 of the 99 infants who completed the study (51.5%) had recurrent wheezing. Respiratory infection was strongly associated with recurrent wheezing (OR 6.67, 95% CI 1.96-22.72), whereas exclusive breastfeeding for at least 1 month was a protective factor (OR 0.09, 95% CI 0.01-0.51). Exposure to high levels of mouse allergen was more frequent among non-recurrent wheezers, approaching significance (OR 0.12, 95% CI 0.01-1.13; p=0.064). None of the children were sensitized to mouse. Sensitization to mite was found in 26/90 (28.8%) children, with no association with recurrent wheezing. Conclusion: Respiratory infection was strongly associated with recurrent wheezing in the first 30 months of life, in children at high risk of asthma, living in a socially deprived community in Brazil. Copyright (C) 2009 S. Karger AG, Basel

A serosurvey for hantavirus infection in wild rodents from the states of Rio de Janeiro and Pernambuco, Brazil

Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported. (C) 2008 Elsevier B.V. All rights reserved.

Oral lesions in HIV infected individuals from Ribeirao Preto, Brazil

Objectives: The aim of this study was to diagnosis oral lesions related to HIV infection in individuals followed in the General Hospital of the School of Medicine of Ribeirao Preto, University of Sao Paulo, Brazil. The presence of oral lesions was correlated with gender, age, smoking habit, levels of CD4 lymphocytes, HIV load, time of HIV seropositivity, AIDS condition, use of removable dental prosthesis, and use of HAART. Materials and Methods: 340 HIV infected individuals were selected for this study, all participants of the study were examined by only one practiced dentist which performed anamnesis, peribuccal and oral examination. Results: Oral lesions were observed in 113 of 340 (33.2%) HIV infected individuals. These oral lesions included: oral candidiasis (17.7%) of pseudomembranous (10.8%) and of erythematous types (6.9%), angular cheilitis (13.9%), hairy leukoplakia (11.8%), and oral ulcers (2.1%). Oral candidiasis lesions were more frequently observed in women (p. 033). Smoking addict participants presented a high frequency of tongue hairy leukoplakia (p. 038) and a reduced frequency of oral ulcers (p. 018). Hairy leukoplakia and pseudomembranous candidiasis were inversely correlated to CD4+ L levels and directly correlated with HIV load, behaving as immune depression markers. Hairy leukoplakia and pseudomembranous candidiasis also showed an inverse correlation with HAART use (p < .0001). Patients using mobile dental prosthesis presented a high frequency of erythematous candidiasis (p. 003). Conclusion: The inverse correlation with CD4+ L level and the direct correlation with HIV load suggest that oral lesions could be used as alternative clinical markers for poor immune condition in HIV infected individuals.

Natural Host Relationships and Genetic Diversity of Rodent-Associated Hantaviruses in Southeastern Brazil

Objective: Hantaviruses are rodent-borne RNA viruses that have caused hantavirus cardiopulmonary syndrome in several Brazilian regions. In the present study, geographical distribution, seroprevalence, natural host range, and phylogenetic relations of rodent-associated hantaviruses collected from seven counties of Southeastern Brazil were evaluated. Methods: ELISA, RT-PCR and phylogenetic analysis were used in this study. Results: Antibodies to hantavirus were detected in Bolomys lasiurus, Akodon sp. and Oligoryzomys sp., performing an overall seroprevalence of 5.17%. All seropositive rodents were associated with grasslands or woods surrounded by sugar cane fields. Phylogenetic analysis of partial S- and M-segment sequences showed that viral sequences isolated from B. lasiurus specimens clustered with Araraquara virus. However, a sequence from Akodon sp. shared 100% similarity with Argentinian/Chilean viruses based on the partial S- segment amino acid sequence. Conclusion: These results indicate that there are associations between rodent reservoirs and hantaviruses in some regions of Southeastern Brazil, and suggest the existence of additional hantavirus genetic diversity and host ecology in these areas. Copyright (C) 2008 S. Karger AG, Basel

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection. (C) 2011 Elsevier B.V. All rights reserved.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

A BALB/c mouse model shows that liver involvement in dengue disease is immune-mediated

In the present study, BALB/c mice were used to develop a model for the hepatic injury associated to dengue infection. Histological analysis after subcutaneous inoculation with a low viral dose of dengue-2 virus showed Kupffer cell hyperplasia and an increased inflammatory cellular infiltrate next to the bile ducts on days 5, 7 and 14 post-inoculation, mainly characterized by the presence of mononuclear cells. The liver mRNA transcription level of IL-1 beta was highest on the 5th day post-infection (p.i.) and decreased by the 21st day, TNF-alpha showed a peak of mRNA transcription after 14 days p.i. coinciding with the regression of cellular infiltrates and elevated expression of TGF-beta mRNA. Serum AST and ALT levels were slightly elevated at 7 and 14 days post-infection. Dengue-2 RNA levels were undetectable in the liver on any of the days following inoculation. Our observations suggest that, as it is true for humans, the animals undergo a transient and slight liver inflammation, probably due to local cytokine production and cellular infiltration in the liver. (C) 2010 Elsevier Inc. All rights reserved.

The H63D genetic variant of the HFE gene is independently associated with the virological response to interferon and ribavirin therapy in chronic hepatitis C

Background Conflicting results have been reported in studies evaluating the relationship between serum markers of iron overload, liver iron deposits, and HFE mutations (C282Y and H63D) in chronic hepatitis C patients, and also their impact on the response to therapy in these patients. Aim To evaluate the role of HFE mutations in the severity of liver disease and in the response to therapy in chronic hepatitis C. Methods Two hundred and sixty-four hepatitis C patients treated with standard interferon and ribavirin were divided into two groups according to type of antiviral response: sustained virological response (SVR) and nonresponse or relapse. We evaluated the relationship between HFE mutation and the type of antiviral response, clinical data, biochemical tests, liver histopathology, virological data, and HFE mutations. Results Of the 264 patients, 88 (32.1%) had SVR whereas 67.9% had nonresponse or relapse. Liver iron deposits were observed in 49.2% of the patients. The factors associated with SVR were hepatitis C virus genotype 2 or 3, transferrin saturation value of 45% or less, and detection of the H63D mutation. HFE mutation was more frequent in patients with iron deposits, but without association with serum iron biochemistry or severity of liver disease. Steatosis was more frequent in patients with liver iron deposits. Conclusion The H63D mutation was an independent factor associated with SVR in chronic hepatitis C patients, as also were hepatitis C virus genotype 2 or 3 and transferrin saturation value of 45% or less. Moreover, the H63D mutation was associated with liver iron deposits. Eur J Gastroenterol Hepatol 22: 1204-1210 (C) 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Oral squamous Cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, all important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also it significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence oil BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC. but not in benign oral lesions.

Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection

Severe dengue infection in humans causes a disease characterized by thrombocytopenia, increased levels of cytokines, increased vascular permeability, hemorrhage, and shock. Treatment is supportive. Activation of platelet-activating factor (PAF) receptor (PAFR) on endothelial cells and leukocytes induces increase in vascular permeability, hypotension, and production of cytokines. We hypothesized that activation of PAFR could account for the major systemic manifestations of dengue infection. Inoculation of adult mice with an adapted strain of Dengue virus caused a systemic disease, with several features of the infection in humans. In PAFR(-/-) mice, there was decreased thrombocytopenia, hemoconcentration, decreased systemic levels of cytokines, and delay of lethality, when compared with WT infected mice. Treatment with UK-74,505, an orally active PAFR antagonist, prevented the above-mentioned manifestations, as well as hypotension and increased vascular permeability, and decreased lethality, even when started 5 days after virus inoculation. Similar results were obtained with a distinct PAFR antagonist, PCA-4246. Despite decreased disease manifestation, viral loads were similar (PAFR(-/-)) or lower (PAFR antagonist) than in WT mice. Thus, activation of PAFR plays a major role in the pathogenesis of experimental dengue infection, and its blockade prevents more severe disease manifestation after infection with no increase in systemic viral titers, suggesting that there is no interference in the ability of the murine host to deal with the infection. PAFR antagonists are disease-modifying agents in experimental dengue infection.

Rhipicephalus (Boophilus) microplus: Clotting time in tick-infested skin varies according to local inflammation and gene expression patterns in tick salivary glands

Ticks deposit saliva at the site of their attachment to a host in order to inhibit haemostasis, inflammation and innate and adaptive immune responses. The anti-haemostatic properties of tick saliva have been described by many studies, but few show that tick infestations or its anti-haemostatic components exert systemic effects in vivo. In the present study, we extended these observations and show that, compared with normal skin, bovine hosts that are genetically susceptible to tick infestations present an increase in the clotting time of blood collected from the immediate vicinity of haemorrhagic feeding pools in skin infested with different developmental stages of Rhipicepahlus microplus; conversely, we determined that clotting time of tick-infested skin from genetically resistant bovines was shorter than that of normal skin. Coagulation and inflammation have many components in common and we determined that in resistant bovines, eosinophils and basophils, which are known to contain tissue factor, are recruited in greater numbers to the inflammatory site of tick bites than in susceptible hosts. Finally, we correlated the observed differences in clotting times with the expression profiles of transcripts for putative anti-haemostatic proteins in different developmental stages of R. microplus fed on genetically susceptible and resistant hosts: we determined that transcripts coding for proteins similar to these molecules are overrepresented in salivary glands from nymphs and males fed on susceptible bovines. Our data indicate that ticks are able to modulate their host`s local haemostatic reactions. In the resistant phenotype, larger amounts of inflammatory cells are recruited and expression of anti-coagulant molecules is decreased tick salivary glands, features that can hamper the tick`s blood meal. (C) 2010 Elsevier Inc. All rights reserved.

Clinical and Immunological Insights on Severe, Adverse Neurotropic and Viscerotropic Disease following 17D Yellow Fever Vaccination

Yellow fever (YF) vaccines (17D-204 and 17DD) are well tolerated and cause very low rates of severe adverse events (YEL-SAE), such as serious allergic reactions, neurotropic adverse diseases (YEL-AND), and viscerotropic diseases (YEL-AVD). Viral and host factors have been postulated to explain the basis of YEL-SAE. However, the mechanisms underlying the occurrence of YEL-SAE remain unknown. The present report provides a detailed immunological analysis of a 23-year-old female patient. The patient developed a suspected case of severe YEL-AVD with encephalitis, as well as with pancreatitis and myositis, following receipt of a 17D-204 YF vaccination. The patient exhibited a decreased level of expression of Fc-gamma R in monocytes (CD16, CD32, and CD64), along with increased levels of NK T cells (an increased CD3(+) CD16(+/-) CD56(+/-)/CD3(+) ratio), activated T cells (CD4(+) and CD8(+) cells), and B lymphocytes. Enhanced levels of plasmatic cytokines (interleukin-6 [IL-6], IL-17, IL-4, IL-5, and IL-10) as well as an exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within NK cells (gamma interferon positive [IFN-gamma(+)], tumor necrosis factor alpha positive [TNF-alpha(+)], and IL-4 positive [IL-4(+)]), CD8(+) T cells (IL-4(+) and IL-5(+)), and B lymphocytes (TNF-alpha(+), IL-4(+), and IL-10(+)). The analysis of CD4(+) T cells revealed a complex profile that consisted of an increased frequency of IL-12(+) and IFN-gamma(+) cells and a decreased percentage of TNF-alpha(+), IL-4(+), and IL-5+ cells. Depressed cytokine synthesis was observed in monocytes (TNF-alpha(+)) following the provision of antigenic stimuli in vitro. These results support the hypothesis that a strong adaptive response and abnormalities in the innate immune system may be involved in the establishment of YEL-AND and YEL-AVD.