Detection of human T-cell lymphotropic virus type 1 in plasma samples

Human T-cell lymphotropic virus type 1 (HTLV-1) is-an RNA virus responsible for diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukemia/lymphoma (ATL). Cell-to-cell contact and Tax-induced clonal expansion of infected cells are the main modes of virus replication, making virus detection during the viremic stage difficult. Consequently, the proviral load is the current virologic marker for disease monitoring, but the mechanisms of progression have not been established yet. Thus, this study investigated the presence of virus in plasma from asymptomatic HTLV-1 carriers and from HAM/TSP patients. Real-time PCR was performed on DNA from 150 plasma samples; 12(8%) had detectable DNA amplification, including 6(4%) asymptomatic HTLV-1 carriers and 14(26%) HAM/TSP patients (p < 0.005). Of the 33 samples submitted for nested PCR, six (18%, p = 0.02) were positive for HTLV-1 RNA in the plasma. Additionally, 26 plasma samples were treated with DNAse enzyme to eliminate any DNA contamination before RNA extraction. Two of them (8%) showed amplification for HTLV-1 (p = 0.5). Therefore, this study described for the first time the detection of free HTLV-1 RNA in plasma from HTLV-1-infected subjects, regardless of their clinical status. Thus, HTLV-1 viral replication does occur in plasma, and other transmission pathways for HTLV-1 should be investigated further. (C) 2011 Elsevier B.V. All rights reserved.

JC virus-associated central nervous system diseases in HIV-infected patients in Brazil: clinical presentations, associated factors with mortality and outcome

Introduction: Several presentations of neurologic complications caused by JC virus (JCV) in human immunodeficiency virus (HIV)-infected patients have been described and need to be distinguished from the "classic" form of progressive multifocal leukoencephalopathy (PML). The objectives of this study were: 1) to describe the spectrum and frequency of presentations of JCV-associated central nervous system (CNS) diseases; 2) identify factors associated with in-hospital mortality of patients with JCV-associated CNS disease; and 3) to estimate the overall mortality of this population. Material and methods: This was a retrospective study of HIV-infected patients admitted consecutively for JCV-associated CNS diseases in a referral teaching center in Sao Paulo, Brazil, from 2002 to 2007. All patients with laboratory confirmed JCV-associated CNS diseases were included using the following criteria: compatible clinical and radiological features associated with the presence of JCV DNA in the cerebrospinal fluid. JCV-associated CNS diseases were classified as follows: 1) classic PML; 2) inflammatory PML; and 3) JC virus granule cell neuronopathy (GCN). Results: We included 47 cases. JCV-associated CNS diseases were classified as follows: 1) classic PML: 42 (89%); 2) inflammatory PML: three (6%); and 3) JC virus GCN: four (9%). Nosocomial pneumonia (p = 0.003), previous diagnosis of HIV infection (p = 0.03), and imaging showing cerebellar and/or brainstem involvement (p = 0.02) were associated with in-hospital mortality. Overall mortality during hospitalization was 34%. Conclusions: Novel presentations of JCV-associated CNS diseases were observed in our setting; nosocomial pneumonia, previous diagnosis of HIV infection, and cerebellar and/or brainstem involvement were associated with in-hospital mortality; and overall mortality was high. (C) 2012 Elsevier Editora Ltda. All rights reserved.

Number of recent sexual partners among blood donors in Brazil: associations with donor demographics, donation characteristics, and infectious disease markers

BACKGROUND: Brazilian blood centers ask candidate blood donors about the number of sexual partners in the past 12 months. Candidates who report a number over the limit are deferred. We studied the implications of this practice on blood safety. STUDY DESIGN AND METHODS: We analyzed demographic characteristics, number of heterosexual partners, and disease marker rates among 689,868 donations from three Brazilian centers between July 2007 and December 2009. Donors were grouped based on maximum number of partners allowed in the past 12 months for each center. Chi-square and logistic regression analysis were conducted to examine associations between demographic characteristics, number of sex partners, and individual and overall positive markers rates for human immunodeficiency virus (HIV), human T-lymphotropic virus Types 1 and 2, hepatitis B virus, hepatitis C virus, and syphilis. RESULTS: First-time, younger, and more educated donors were associated with a higher number of recent sexual partners, as was male sex in Sao Paulo and Recife (p < 0.001). Serologic markers for HIV and syphilis and overall were associated with multiple partners in Sao Paulo and Recife (p < 0.001), but not in Belo Horizonte (p = 0.05, p = 0.94, and p = 0.75, respectively). In logistic regression analysis, number of recent sexual partners was associated with positive serologic markers (adjusted odds ratio [AOR], 1.2-1.5), especially HIV (AOR, 1.9-4.4). CONCLUSIONS: Number of recent heterosexual partners was associated with HIV positivity and overall rates of serologic markers of sexually transmitted infections. The association was not consistent across centers, making it difficult to define the best cutoff value. These findings suggest the use of recent heterosexual contacts as a potentially important deferral criterion to improve blood safety in Brazil.

Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the non-inoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

First report of sacbrood virus in honey bee (Apis mellifera) colonies in Brazil

Sacbrood disease, an affliction of honey bees (Apis mellifera) characterized by brood that fails to pupate and subsequently dies, is an important threat to honey bee health. The disease is caused by the sacbrood virus (SBV), a positive-, single-stranded RNA virus in the order Picornavirales. Because of the economic importance of honey bees for both pollination and honey production, it is vital to understand and monitor the spread of viruses such as SBV. This virus has been found in many places across the globe, including recently in some South American countries, and it is likely that it will continue to spread. We performed a preliminary study to search for SBV in two apiaries of Africanized honey bees in the State of Sao Paulo, Brazil, using RT-PCR and Sanger sequencing and found the first evidence of SBV in honey bee colonies in Brazil. The virus was detected in larvae, foraging and nurse bees from two colonies, one of which had symptoms of sacbrood disease, at the beginning of the winter season in June 2011. No SBV was found in samples from nine other nearby colonies.

Mayaro Virus Infection in Amazonia: A Multimodel Inference Approach to Risk Factor Assessment

Background: Arboviral diseases are major global public health threats. Yet, our understanding of infection risk factors is, with a few exceptions, considerably limited. A crucial shortcoming is the widespread use of analytical methods generally not suited for observational data - particularly null hypothesis-testing (NHT) and step-wise regression (SWR). Using Mayaro virus (MAYV) as a case study, here we compare information theory-based multimodel inference (MMI) with conventional analyses for arboviral infection risk factor assessment. Methodology/Principal Findings: A cross-sectional survey of anti-MAYV antibodies revealed 44% prevalence (n = 270 subjects) in a central Amazon rural settlement. NHT suggested that residents of village-like household clusters and those using closed toilet/latrines were at higher risk, while living in non-village-like areas, using bednets, and owning fowl, pigs or dogs were protective. The "minimum adequate" SWR model retained only residence area and bednet use. Using MMI, we identified relevant covariates, quantified their relative importance, and estimated effect-sizes (beta +/- SE) on which to base inference. Residence area (beta(Village) = 2.93 +/- 0.41; beta(Upland) = -0.56 +/- 0.33, beta(Riverbanks) = -2.37 +/- 0.55) and bednet use (beta = -0.95 +/- 0.28) were the most important factors, followed by crop-plot ownership (beta = 0.39 +/- 0.22) and regular use of a closed toilet/latrine (beta = 0.19 +/- 0.13); domestic animals had insignificant protective effects and were relatively unimportant. The SWR model ranked fifth among the 128 models in the final MMI set. Conclusions/Significance: Our analyses illustrate how MMI can enhance inference on infection risk factors when compared with NHT or SWR. MMI indicates that forest crop-plot workers are likely exposed to typical MAYV cycles maintained by diurnal, forest dwelling vectors; however, MAYV might also be circulating in nocturnal, domestic-peridomestic cycles in village-like areas. This suggests either a vector shift (synanthropic mosquitoes vectoring MAYV) or a habitat/habits shift (classical MAYV vectors adapting to densely populated landscapes and nocturnal biting); any such ecological/adaptive novelty could increase the likelihood of MAYV emergence in Amazonia.

High Rates of Detection of Respiratory Viruses in Tonsillar Tissues from Children with Chronic Adenotonsillar Disease

Chronic tonsillar diseases are an important health problem, leading to large numbers of surgical procedures worldwide. Little is known about pathogenesis of these diseases. In order to investigate the role of respiratory viruses in chronic adenotonsillar diseases, we developed a cross-sectional study to determine the rates of viral detections of common respiratory viruses detected by TaqMan real time PCR (qPCR) in nasopharyngeal secretions, tonsillar tissues and peripheral blood from 121 children with chronic tonsillar diseases, without symptoms of acute respiratory infections. At least one respiratory virus was detected in 97.5% of patients. The viral co-infection rate was 69.5%. The most frequently detected viruses were human adenovirus in 47.1%, human enterovirus in 40.5%, human rhinovirus in 38%, human bocavirus in 29.8%, human metapneumovirus in 17.4% and human respiratory syncytial virus in 15.7%. Results of qPCR varied widely between sample sites: human adenovirus, human bocavirus and human enterovirus were predominantly detected in tissues, while human rhinovirus was more frequently detected in secretions. Rates of virus detection were remarkably high in tonsil tissues: over 85% in adenoids and close to 70% in palatine tonsils. In addition, overall virus detection rates were higher in more hypertrophic than in smaller adenoids (p = 0.05), and in the particular case of human enteroviruses, they were detected more frequently (p = 0.05) in larger palatine tonsils than in smaller ones. While persistence/latency of DNA viruses in tonsillar tissues has been documented, such is not the case of RNA viruses. Respiratory viruses are highly prevalent in adenoids and palatine tonsils of patients with chronic tonsillar diseases, and persistence of these viruses in tonsils may stimulate chronic inflammation and play a role in the pathogenesis of these diseases.

In vitro expression and antiserum production against the movement protein of Citrus leprosis virus C (CiLV-C)

Citrus leprosis, caused by Citrus leprosis virus C (CiLV-C), is currently considered the most important viral disease in the Brazilian citrus industry due to the high costs required for the chemical control of its vector, the mite Brevipalpus phoenicis. The pathogen induces a non-systemic infection and the disease is characterized by the appearance of localized lesions on citrus leaves, stems and fruits, premature fruit and leaf drop and dieback of stems. Attempts were made to promote in vitro expression of the putative cell-to-cell movement protein of CiLV-C in Escherichia coli and to produce a specific polyclonal antibody against this protein as a tool to investigate the virus-plant-vector relationship. The antibody reacted strongly with the homologous protein expressed in vitro by ELISA, but poorly with the native protein present in leaf lesion extracts from sweet orange caused by CiLV-C. Reactions from old lesions were more intense than those from young lesions. Western blot and in situ immunolocalization assays failed to detect the native protein. These results suggest low expression of the movement protein (MP) in host tissues. Moreover, it is possible that the conformation of the protein expressed in vitro and used to produce the antibody differs from that of the native MP, hindering a full recognition of the latter.

Immunogenicity of a meningococcal serogroup C conjugate vaccine in HIV-infected children, adolescents, and young adults

Children and adolescents infected with HIV typically have a lower response to immunization than do those in the general population. In most developed countries, meningococcal serogroup C conjugate vaccine is one of the recommended vaccines for such individuals. However, there have been no studies evaluating the antibody response to this vaccine in HIV-infected children, adolescents or young adults. In this study, we evaluated that response using serum bactericidal antibody (SBA) and enzyme-linked immunosorbent assay, comparing HIV-infected with non-HIV-infected patients, as well as analysing the occurrence of side effects. In non-responders, we assessed the antibody response to revaccination. This clinical trial involved 92 patients between 10 and 20 years of age: 43 HIV-infected patients (HIV+ group) and 49 non-HIV-infected patients (HIV- group). After one dose of the vaccine, 72.1% of the HIV+ group patients and 100% of the HIV- group patients were considered protected. Of the HIV+ group patients who received a second dose of the vaccine, only 40% acquired protection. Overall, 81.4% of the HIV+ group patients acquired protection (after one or two doses of the vaccine). Side effects occurred in 16.3% and 44% of the HIV+ group and HIV- group patients, respectively. Therefore, the meningococcal serogroup C conjugate vaccine proved to be safe and effective for use in HIV-infected children, adolescents, and young adults, although their antibody response was weaker than that shown by non-HIV-infected patients. This indicates the need to discuss changes to the immunization schedule for children, adolescents, and young adults infected with HIV, in order to ensure more effective protection against meningococcal disease. (c) 2012 Elsevier Ltd. All rights reserved.

Immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus

Objectives The aim of the present paper is to evaluate the immune response and tolerability of varicella vaccine in children and adolescents with systemic lupus erythematosus previously exposed to varicella-zoster virus. Methods We performed a prospective and controlled study on a group of 54 SLE patients that were chosen at random to be or not to be vaccinated (28 were vaccinated and 26 were not). Twenty-eight healthy controls, of matching age and sex were also vaccinated. All were submitted to a questionnaire, physical evaluation and laboratory assays: lymphocyte immuno-phenotyping by flow cytometry, plasma varicella zoster virus (VZV) serology by ELISA and in vitro interferon gamma (IFN gamma) production by T-cells after stimulus with VZV antigen. They were evaluated before vaccination and at 30, 45, 180 and 360 days afterwards. Results We did not observe any differences in the frequency of adverse events in both vaccinated groups. At study entry, all individuals were seropositive for VZV antibodies. The serum VZV antibody titres similarly increased after vaccination. The frequency of flares and the SLEDAI score were also similar among the patients. Thirty days after vaccination the production of IFN gamma specific to VZV was lower in the SLE group compared to healthy, controls. In the follow-up we observed 4 cases of herpes zoster in the SLE unvaccinated group, but no zoster in the vaccinated group. Conclusion The varicella vaccine was well tolerated in SLE group, who had pre-existing immunity to varicella. The varicella vaccine immunogenicity measurement by serum antibody titres was appropriate. The incidence of HZ was lower in the vaccinated lupus group.

Motor Neuron Disease and Acquired Axonal Neuropathy Association in HIV Infection: Case Report and Update

Background: A possible viral etiology has been documented in the genesis of motor neuron disorders and acquired peripheral neuropathies, mainly due to the vulnerability of peripheral nerves and the anterior horn to certain viruses. In recent years, several reports show association of HIV infection with Amyotrophic Lateral Sclerosis Syndrome, Motor Neuron Diseases and peripheral neuropathies. Objective: To report a case of an association between Motor Neuron Disease and Acquired Axonal neuropathy in HIV infection, and describe the findings of neurological examination, cerebrospinal fluid, neuroimaging and electrophysiology. Methods: The patient underwent neurological examination. General medical examinations were performed, including, specific neuromuscular tests, analysis of cerebrospinal fluid, muscle biopsy and imaging studies. Results and Discussion: The initial clinical presentation of our case was marked by cramps and fasciculations with posterior distal paresis and atrophy in the left arm. We found electromyography tracings with deficits in the anterior horn of the spinal cord and peripheral nerves. Dysphagia and release of primitive reflexes were also identified. At the same time, the patient was informed to be HIV positive with high viral load. He received antiretroviral therapy, with load control but with no clinical remission. Conclusion: Motor Neuron disorders and peripheral neuropathy may occur in association with HIV infection. However, a causal relationship remains uncertain. It is noteworthy that the antiretroviral regimen may be implicated in some cases.

Faster HIV-1 Disease Progression among Brazilian Individuals Recently Infected with CXCR4-Utilizing Strains

Introduction: Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression. Methods: A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/mu L. Results: Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4,350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4.350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4.350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype. Conclusions: Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.

Interleukin-18 and interferon-gamma polymorphisms are implicated on proviral load and susceptibility to human T-lymphotropic virus type 1 infection

Interleukin-18 (IL-18) and interferon-gamma (IFN-?) exert important functions in both innate and adaptive immune responses against intracellular pathogens and viruses. Previous studies suggested that host genetic factors, including cytokines gene polymorphisms, could be involved in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Thus, we analyzed -137C/G and -607A/C of the IL-18 promoter and +874T/A of the IFN-? in DNA samples from 98 HTLV-1-infected individuals exhibiting or not clinical symptoms and 150 healthy control individuals. The IL-18 promoter -607CC genotype was significantly lower in HTLV-1 asymptomatic carriers (HAC) and HTLV-1-infected individuals (HAC + HAM/TSP) than healthy control group. In contrast, the -607AC genotype was significantly higher in HAC and HTLV-1-infected individuals group compared to the healthy control group. The -137G/-607A IL-18 haplotype was higher in infected group than healthy control group, and the -137C/-607C IL-18 haplotype was increased in the healthy control group compared to the others. Finally, the IFN-? polymorphism analysis showed that the HTLV-1-infected individuals with +874AT genotype presented higher proviral load than +874AA genotype. These data indicate that the IL-18-607AC genotype and -137G/-607A haplotype could be a risk factor for HTLV-1 infection, whereas the protective effect could be conferred by -607CC genotype and -137C/-607C haplotype. Also, the IFN-? could be implicated on the proviral load levels.

Low Sensitivity of NS1 Protein Tests Evidenced during a Dengue Type 2 Virus Outbreak in Santos, Brazil, in 2010

In 2010, a large outbreak of dengue occurred in Santos, Brazil. The detection of the NS1 antigen was used for diagnosis in addition to the detection of IgG, IgM, and RNA. A large number of NS1 false-negative results were obtained. A total of 379 RNA-positive samples were selected for thorough evaluation. NS1 was reactive in 37.7% of cases. Most of the cases were characterized as a secondary infection by dengue 2 virus. Sequencing of NS1 positive and negative isolates did not reveal any mutation that could justify the diagnostic failure. Use of existing NS1 tests in the Brazilian population may present a low negative predictive value, and they should be used with caution, preferentially after performing a validation with samples freshly obtained during the ongoing epidemic.

Crotoxin and phospholipases A(2) from Crotalus durissus terrificus showed antiviral activity against dengue and yellow fever viruses

Dengue is the most important arbovirus in the world with an estimated of 50 million dengue infections occurring annually and approximately 2.5 billion people living in dengue endemic countries. Yellow fever is a viral hemorrhagic fever with high mortality that is transmitted by mosquitoes. Effective vaccines against yellow fever have been available for almost 70 years and are responsible for a significant reduction of occurrences of the disease worldwide; however, approximately 200,000 cases of yellow fever still occur annually, principally in Africa. Therefore, it is a public health priority to develop antiviral agents for treatment of these virus infections. Crotalus durissus terrificus snake, a South American rattlesnake, presents venom with several biologically actives molecules. In this study, we evaluated the antiviral activity of crude venom and isolated toxins from Crotalus durissus terrificus and found that phospholipases A(2) showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells. (C) 2011 Elsevier Ltd. All rights reserved.