Detection of dengue virus in saliva and urine by real time RT-PCR

Early diagnosis of dengue virus (DENV) infection is important for patient management and control of dengue outbreaks. The objective of this study was to analyze the usefulness of urine and saliva samples for early diagnosis of DENV infection by real time RT-PCR. Two febrile patients, who have been attended at the General Hospital of the School of Medicine of Ribeirao Preto, Sao Paulo University were included in the study. Serum, urine and saliva samples collected from both patients were subjected to real time RT-PCR for DENV detection and quantification. Dengue RNA was detected in serum, urine and saliva samples of both patients. Patient 1 was infected with DENV-2 and patient 2 with DENV-3. Data presented in this study suggest that urine and saliva could be used as alternative samples for early diagnosis of dengue virus infection when blood samples are difficult to obtain, e.g.,in newborns and patients with hemorrhagic syndromes.

Hantavirus Pulmonary Syndrome, Central Plateau, Southeastern, and Southern Brazil

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HIPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent lung tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.

Low- and High-Intensity Lasers in the Treatment of Herpes Simplex Virus 1 Infection

Herpes simplex virus (HSV) is one of the most common viral infections of the human being. Although most of the seropositive persons do not manifest symptoms, infected individuals may present recurrent infections, characterized by cold sores. HSV-1 infection can result in potentially harmful complications in some patients, especially in those with compromised immunity. We report a clinical case of a patient with severe oral HSV-1 infection in the lower lip. The treatment of the lesions with the association of high-intensity (erbium-doped yttrium aluminum garnet, 2.94 mu m, 80 mJ/pulse, 2-4 Hz) and low-intensity (indium gallium aluminum phosphide, 660 nm, 3.8 J/cm(2), 10mW) lasers has not been reported in the literature. During treatment, no systemic or topical medication was used. Pain sensitivity was completely gone after the first irradiation with the low-intensity laser. During the healing process, lesions were traumatized twice, on the days 4 and 7. Even though the lesions were completely healed within 10 days.

Yellow Fever Virus in Haemagogus leucocelaenus and Aedes serratus Mosquitoes, Southern Brazil, 2008

Yellow fever virus (YFV) was isolated from Haemagogus leucocelaenus mosquitoes during an epizootic in 2001 in the Rio Grande do Sul State in southern Brazil In October 2008 a yellow fever outbreak was reported there with nonhuman primate deaths and human cases This latter outbreak led to intensification of surveillance measures for early detection of YFV and support for vaccination programs We report entomologic surveillance in 2 municipalities that recorded nonhuman primate deaths Mosquitoes were collected at ground level identified and processed for virus isolation and molecular analyses Eight YFV strains were isolated (7 from pools of Hg leucocelaenus mosquitoes and another from Aedes serratus mosquitoes) 6 were sequenced and they grouped in the YFV South American genotype I The results confirmed the role of Hg leucocelaenus mosquitoes as the main YFV vector in southern Brazil and suggest that Ae serratus mosquitoes may have a potential role as a secondary vector

Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a ""flipflop'' phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in Sao Paulo state, Brazil. We show that different viral genotypes entered Sao Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in Sao Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.

Var transcription profiling of Plasmodium falciparum 3D7: assignment of cytoadherent phenotypes to dominant transcripts

Background: Cytoadherence of Plasmodium falciparum-infected red blood cells is mediated by var gene-encoded P. falciparum erythrocyte membrane protein-1 and host receptor preference depends in most cases on which of the 50-60 var genes per genome is expressed. Enrichment of phenotypically homogenous parasites by panning on receptor expressing cells is fundamental for the identification of the corresponding var transcript. Methods: P. falciparum 3D7 parasites were panned on several transfected CHO-cell lines and their var transcripts analysed by i) reverse transcription/PCR/cloning/sequencing using a universal DBL alpha specific oligonucleotide pair and ii) by reverse transcription followed by quantitative PCR using 57 different oligonucleotide pairs. Results: Each cytoadherence selected parasite line also adhered to untransfected CHO-745 cells and upregulation of the var gene PFD995/PFD1000c was consistently associated with cytoadherence to all but one CHO cell line. In addition, parasites panned on different CHO cell lines revealed candidate var genes which reproducibly associated to the respective cytoadherent phenotype. The transcription profile obtained by RT-PCR/cloning/sequencing differed significantly from that of RT-quantitative PCR. Conclusion: Transfected CHO cell lines are of limited use for the creation of monophenotypic cytoadherent parasite lines. Nevertheless, 3D7 parasites can be reproducibly selected for the transcription of different determined var genes without genetic manipulation. Most importantly, var transcription analysis by RT-PCR/cloning/sequencing may lead to erroneous interpretation of var transcription profiles.

Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

Background: Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45) is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF) is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods: In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-kappa B activation was also determined by electrophoretic mobility shift assay (EMSA) using specific oligonucleotides and nuclear protein extracts. Results: We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7), SOD2 (superoxide dismutase 2), 100P (S100 calcium binding protein P), PI3 (protease inhibitor 3, skin-derived), CSTA (cystatin A), RARRES1 (retinoic acid receptor responder 1), and LXN (latexin). The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-kappa B activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion: This is the first in depth analysis of the differential effect of TNF on normal and HPV16 or HPV18 immortalized keratinocytes. Our findings may be useful for the identification of genes involved in TNF resistance acquisition and candidate genes which deregulated expression may be associated with cervical disease establishment and/or progression.

Intercellular transport of epidermis-expressed MADS domain transcription factors and their effect on plant morphology and floral transition

P>During the lifetime of an angiosperm plant various important processes such as floral transition, specification of floral organ identity and floral determinacy, are controlled by members of the MADS domain transcription factor family. To investigate the possible non-cell-autonomous function of MADS domain proteins, we expressed GFP-tagged clones of AGAMOUS (AG), APETALA3 (AP3), PISTILLATA (PI) and SEPALLATA3 (SEP3) under the control of the MERISTEMLAYER1 promoter in Arabidopsis thaliana plants. Morphological analyses revealed that epidermal overexpression was sufficient for homeotic changes in floral organs, but that it did not result in early flowering or terminal flower phenotypes that are associated with constitutive overexpression of these proteins. Localisations of the tagged proteins in these plants were analysed with confocal laser scanning microscopy in leaf tissue, inflorescence meristems and floral meristems. We demonstrated that only AG is able to move via secondary plasmodesmata from the epidermal cell layer to the subepidermal cell layer in the floral meristem and to a lesser extent in the inflorescence meristem. To study the homeotic effects in more detail, the capacity of trafficking AG to complement the ag mutant phenotype was compared with the capacity of the non-inwards-moving AP3 protein to complement the ap3 mutant phenotype. While epidermal expression of AG gave full complementation, AP3 appeared not to be able to drive all homeotic functions from the epidermis, perhaps reflecting the difference in mobility of these proteins.

Recombinant rabies virus glycoprotein synthesis in bioreactor by transfected Drosophila melanogaster S2 cells carrying a constitutive or an inducible promoter

S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 degrees C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained similar to 1.5-3 x 10(7) cells/mL after 3-4 clays of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 mu g/10(7) cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO(4) induction attaining specific productivities of 1.5-2 mu g/10(7) cells at clays 4-5. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 mu g/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.5-3 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function. (C) 2010 Elsevier B.V. All rights reserved.

Characterization and Experimental Host Range of a Brazilian Tomato Isolate of Tomato severe rugose virus

We report the complete molecular characterization of the DNA-A and DNA-B of a Brazilian tomato isolate of Tomato severe rugose virus (ToSRV) and the experimental host range of the virus determined using white-fly transmission tests. Genome analysis showed that ToSRV has a close evolutionary relationship with Tomato rugose mosaic virus. Of 33 plants species inoculated with viruliferous Bemisia tabaci biotype B, 13 species were susceptible to ToSRV, nine asymptomatically. Therefore, ToSRV disease management strategy should include the control of infected weeds close to tomato fields.

Natural infection of Nicandra physaloides by Tomato severe rugose virus in Brazil

Nicandra physaloides, a common weed in South America, was found to be infected by an isolate of Tomato severe rugose virus (ToSRV), a bipartite begomovirus. The plants developed severe yellow rugose mosaic and were collected in So Paulo State, Brazil. This isolate of ToSRV was transmitted by Bemisia tabaci B biotype from infected plants of N. physaloides to healthy plants of N. physaloides and tomato in a glasshouse. This is the first report of natural infection of N. physaloides by ToSRV in Brazil.

Transgenic Sweet Orange (Citrus sinensis L. Osbeck) Expressing the attacin A Gene for Resistance to Xanthomonas citri subsp citri

Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and spray-inoculated with an Xcc suspension (10(6) cfu mL(-1)). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (non-transgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were `Caipira` sweet orange (Citrus sinensis L. Osbeck) + `Rangpur` lime (C. limonia Osbeck), `Caipira` sweet orange + `Cleopatra` mandarin (C. reshni hort. ex Tanaka), `Caipira` sweet orange + `Volkamer` lemon (C. volkameriana V Ten. & Pasq.), `Caipira` sweet orange + rough lemon (C. jambhiri Lush.), `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange (C. aurantium L.), `Rangpur` lime + `Sunki` mandarin (C. sunki (Hayata) hort. ex Tanaka), `Ruby Blood` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, `Rohde Red` sweet orange (C. sinensis L. Osbeck) + `Volkamer` lemon, and `Valencia` sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. `Cleopatra` mandarin + sour orange, `Rangpur` lime + `Sunki` mandarin, `Cleopatra` mandarin + `Volkamer` lemon, `Ruby Blood` sweet orange + `Volkamer` lemon, `Rohde Red` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Volkamer` lemon had less trunk rot occurrence, whereas the somatic hybrids `Cleopatra` mandarin + `Volkamer` lemon, `Cleopatra` mandarin + sour orange, `Caipira` sweet orange + `Volkamer` lemon, and `Caipira` sweet orange + `Rangpur` lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy `Valencia` sweet orange budwood. Differences in average scion shoot length indicated that the hybrids `Cleopatra` mandarin + sour orange and `Valencia` sweet orange + Fortunella obovata were intolerant to CTV (c) 2007 Elsevier B.V. All rights reserved.

Genetic Transformation of Passionflower and Evaluation of R(1) and R(2) Generations for Resistance to Cowpea aphid borne mosaic virus

We report on the production and evaluation of passionflower transgenic lines for resistance to Cowpea aphid borne mosaic virus (CABMV). Genetic transformation was done using Agrobacterium tumefaciens and transgene integration was confirmed by Southern blot analyses, resulting in nine transgenic lines for `IAC 275` and three for `IAC 277`. Transgenic lines were clonally propagated and evaluated for resistance to CABMV After the third inoculation, under higher inoculum pressure, only propagated plants of the transgenic line T16 remained asymptomatic, indicating a high resistance to infection with CABMV. This transgenic line was self-pollinated and the RI generation was evaluated together with the RI generation of another resistant transgenic line (T2) identified previously. Plants were inoculated with CABMV by means of viruliferous Myzus nicotianae. All 524 T2R(1) plants became infected, whereas 13 of 279 T16R(1) remained asymptomatic after four successive inoculations. A TI6R(2) generation was obtained and plants were inoculated with CABMV mechanically or by aphids. After successive inoculations, 118 of 258 plants were symptomless, suggesting that the resistance to CABMV was maintained in the plant genome as the homozygous condition was achieved. Five selected resistant TI6R(2) plants which contained the capsid protein gene are being crossed for further analyses.