Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Viruses face a double defense by plant small RNAs

Plants fight viral infections with enzymes that digest viral RNA, but viruses retaliate with proteins that suppress these enzymes. To boost their antiviral response plants deploy enzymes with redundant functions.

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway. © 2008 Federation of European Biochemical Societies.

Characterisation of the subgenomic RNAs of an Australian isolate of barley yellow dwarf luteovirus

We have characterised the subgenomic RNAs of an Australian isolate of BYDV-PAV. Northern blot analyses of infected plants and protoplasts have shown that this isolate synthesises three subgenomic RNAs. Precise mapping of the transcription start sites of all three subgenomic RNAs and translational analyses of subgenomic RNA 2 and 3 have revealed a number of features. First, the transcription start site of subgenomic RNA 1 in this isolate differs markedly from the start site determined for an Illinois isolate of BYDV-PAV. Second, the start sites of subgenomic RNA 1 and 2 occur at a sequence that closely resembles the 5' end sequence of the genomic RNA (5'AGUGAAGA). Third, subgenomic RNA 2 appears to express ORF 6 of BYDV-PAV but the gene product is truncated due to the appearance of a new stop codon in the sequence. Last, subgenomic RNA 3, which is abundantly transcribed and encapsidated by the virus particle, appears to have no coding ability. We postulate that this novel subgenomic RNA has a regulatory function.

Carrot red leaf and carrot mottle viruses : observations on the composition of the particles in single and mixed infections

Particles of carrot red leaf virus (CRLV; luteovirus group) purified from chervil (Anthriscus cerefolium) contain a single ssRNA species of mol. wt. about 1.8 x 106 and a major protein of mol. wt. about 25000. CRLV acts as a helper for aphid transmission of carrot mottle virus (CMotV; ungrouped) from mixedly infected plants. Virus preparations purified from such plants possess the infectivity of both viruses but contain particles indistinguishable from those of CRLV; some of the particles are therefore thought to consist of CMotV RNA packaged in CRLV coat protein. When RNA from such preparations was electrophoresed in agarose/polyacrylamide gels, CMotV infectivity was associated with an RNA band that migrated ahead of the CRLV RNA band and had an estimated mol. wt. of about 1.5 x 106, similar to that previously found for the infective ssRNA extracted directly from Nicotiana clevelandii leaves infected with CMotV alone. Preparations of dsRNA from CMotV-infected N. clevelandii leaves contained two species: one of mol. wt. about 3.2 x 106, presumably the replicative form of the infective ssRNA, and the other, mol. wt. about 0.9 x 106, of unknown origin and function. The infective agent in buffer extracts of CMotV-infected N. clevelandii was resistant to RNase (although the enzyme acted as a reversible inhibitor of infection at high concentrations) and is therefore not unprotected RNA. It may be protected within the approximately 52 nm enveloped structures previously reported.

Evolutionary relationships between bat coronaviruses and their hosts

Recent studies have suggested that bats are the natural reservoir of a range of coronaviruses (CoVs), and that rhinolophid bats harbor viruses closely related to the severe acute respiratory syndrome (SARS) CoV, which caused an outbreak of respiratory illness in humans during 2002-2003. We examined the evolutionary relationships between bat CoVs and their hosts by using sequence data of the virus RNA-dependent RNA polymerase gene and the bat cytochrome b gene. Phylogenetic analyses showed multiple incongruent associations between the phylogenies of rhinolophid bats and their CoVs, which suggested that host shifts have occurred in the recent evolutionary history of this group. These shifts may be due to either virus biologic traits or host behavioral traits. This finding has implications for the emergence of SARS and for the potential future emergence of SARS-CoVs or related viruses.

The Structure and Functions of Hantavirus Nucleocapsid Protein

Hantaviruses, members of the genus Hantavirus in the Bunyaviridae family, are enveloped single-stranded RNA viruses with tri-segmented genome of negative polarity. In humans, hantaviruses cause two diseases, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS), which vary in severity depending on the causative agent. Each hantavirus is carried by a specific rodent host and is transmitted to humans through excreta of infected rodents. The genome of hantaviruses encodes four structural proteins: the nucleocapsid protein (N), the glycoproteins (Gn and Gc), and the polymerase (L) and also the nonstructural protein (NSs). This thesis deals with the functional characterization of hantavirus N protein with regard to its structure. Structural studies of the N protein have progressed slowly and the crystal structure of the whole protein is still not available, therefore biochemical assays coupled with bioinformatical modeling proved essential for studying N protein structure and functions. Presumably, during RNA encapsidation, the N protein first forms intermediate trimers and then oligomers. First, we investigated the role of N-terminal domain in the N protein oligomerization. The results suggested that the N-terminal region of the N protein forms a coiled-coil, in which two antiparallel alpha helices interact via their hydrophobic seams. Hydrophobic residues L4, I11, L18, L25 and V32 in the first helix and L44, V51, L58 and L65 in the second helix were crucial for stabilizing the structure. The results were consistent with the head-to-head, tail-to-tail model for hantavirus N protein trimerization. We demonstrated that an intact coiled-coil structure of the N terminus is crucial for the oligomerization capacity of the N protein. We also added new details to the head-to-head, tail-to-tail model of trimerization by suggesting that the initial step is based on interaction(s) between intact intra-molecular coiled-coils of the monomers. We further analyzed the importance of charged aa residues located within the coiled-coil for the N protein oligomerization. To predict the interacting surfaces of the monomers we used an upgraded in silico model of the coiled-coil domain that was docked into a trimer. Next the predicted target residues were mutated. The results obtained using the mammalian two-hybrid assay suggested that conserved charged aa residues within the coiled-coil make a substantial contribution to the N protein oligomerization. This contribution probably involves the formation of interacting surfaces of the N monomers and also stabilization of the coiled-coil via intramolecular ionic bridging. We proposed that the tips of the coiled-coils are the first to come into direct contact and thus initiate tight packing of the three monomers into a compact structure. This was in agreement with the previous results showing that an increase in ionic strength abolished the interaction between N protein molecules. We also showed that residues having the strongest effect on the N protein oligomerization are not scattered randomly throughout the coiled-coil 3D model structure, but form clusters. Next we found evidence for the hantaviral N protein interaction with the cytoplasmic tail of the glycoprotein Gn. In order to study this interaction we used the GST pull-down assay in combination with mutagenesis technique. The results demonstrated that intact, properly folded zinc fingers of the Gn protein cytoplasmic tail as well as the middle domain of the N protein (that includes aa residues 80 248 and supposedly carries the RNA-binding domain) are essential for the interaction. Since hantaviruses do not have a matrix protein that mediates the packaging of the viral RNA in other negatve stranded viruses (NSRV), hantaviral RNPs should be involved in a direct interaction with the intraviral domains of the envelope-embedded glycoproteins. By showing the N-Gn interaction we provided the evidence for one of the crucial steps in the virus replication at which RNPs are directed to the site of the virus assembly. Finally we started analysis of the N protein RNA-binding region, which is supposedly located in the middle domain of the N protein molecule. We developed a model for the initial step of RNA-binding by the hantaviral N protein. We hypothesized that the hantaviral N protein possesses two secondary structure elements that initiate the RNA encapsidation. The results suggest that amino acid residues (172-176) presumably act as a hook to catch vRNA and that the positively charged interaction surface (aa residues 144-160) enhances the initial N-RNA interacation. In conclusion, we elucidated new functions of hantavirus N protein. Using in silico modeling we predicted the domain structure of the protein and using experimental techniques showed that each domain is responsible for executing certain function(s). We showed that intact N terminal coiled-coil domain is crucial for oligomerization and charged residues located on its surface form a interaction surface for the N monomers. The middle domain is essential for interaction with the cytoplasmic tail of the Gn protein and RNA binding.

Immunovirological Correlates in Human Rabies Treated With Therapeutic Coma

A 37-year-old woman was admitted to hospital and over the next 5 days developed a progressive encephalitis. Nuchal skin biopsy, analyzed using a Rabies TaqMan(C) PCR, demonstrated rabies virus RNA. She had a history in keeping with exposure to rabies whilst in South Africa, but had not received pre- or post-exposure prophylaxis. She was treated with a therapeutic coma according to the

False-positive PCR results linked to administration of seasonal influenza vaccine

False-positive PCR results usually occur as a consequence of specimen-to-specimen or amplicon-to-specimen contamination within the laboratory. Evidence of contamination at time of specimen collection linked to influenza vaccine administration in the same location as influenza sampling is described. Clinical, circumstantial and laboratory evidence was gathered for each of five cases of influenza-like illness (ILI) with unusual patterns of PCR reactivity for seasonal H1N1, H3N2, H1N1 (2009) and influenza B viruses. Two 2010 trivalent influenza vaccines and environmental swabs of a hospital influenza vaccination room were also tested for influenza RNA. Sequencing of influenza A matrix (M) gene amplicons from the five cases and vaccines was undertaken. Four 2009 general practitioner (GP) specimens were seasonal H1N1, H3N2 and influenza B PCR positive. One 2010 GP specimen was H1N1 (2009), H3N2 and influenza B positive. PCR of 2010 trivalent vaccines showed high loads of detectable influenza A and B RNA. Sequencing of the five specimens and vaccines showed greatest homology with the M gene sequence of Influenza A/Puerto Rico/8/1934 H1N1 virus (used in generation of influenza vaccine strains). Environmental swabs had detectable influenza A and B RNA. RNA detection studies demonstrated vaccine RNA still detectable for at least 66 days. Administration of influenza vaccines and clinical sampling in the same room resulted in the contamination with vaccine strains of surveillance swabs collected from patients with ILI. Vaccine contamination should therefore be considered, particularly where multiple influenza virus RNA PCR positive signals (e.g. H1N1, H3N2 and influenza B) are detected in the same specimen.

The results of a randomized trial looking at 24 weeks vs 48 weeks of treatment with peginterferon alpha-2a (40 kDa) and ribavirin combination therapy in patients with chronic hepatitis C genotype 1

Peginterferon-alpha plus ribavirin is the most effective therapy for chronic hepatitis C. This study was designed to evaluate the effect of peginterferon alpha-2a (40 kDa) plus ribavirin on sustained virological response (SVR) when administered for 24 vs 48 weeks in genotype 1 naive patients. One hundred and seventeen patients were enrolled in this controlled trial. Genotype 1 patients were randomized to 24 weeks treatment vs 48 weeks treatment. Genotype non-1 patients received 24 weeks treatment as an observational group. Outcomes were SVR (defined by hepatitis C virus-RNA-negative at week 24 of follow-up) and tolerability across the study period. The end-of-treatment response was 59% for genotype 1 (24 weeks treatment), 80% for genotype 1 (48 weeks treatment) and 92% for genotype non-1 (24 weeks treatment). The end-of-follow-up response was 19% (95% confidence interval (CI): 7.2-36.4) (genotype 1, 24 weeks) and 48% (95% CI: 30.2-66.9; P = 0.0175) (genotype 1, 48 weeks). Among genotype non-1, SVR was 76% (95% CI: 62.3-86.5). There were no unexpected adverse events.Almost half of the genotype 1 patients achieved an SVR after 48 weeks treatment with peginterferon alpha-2a (40 kDa) and low-dose ribavirin and confirmed that they should be treated for 48 weeks. Safety profile was acceptable.

Induction pegylated interferon Alfa-2a and high dose ribavirin do not increase SVR in heavy patients with HCV genotype 1 and high viral loads

Background & Aims Patients infected with hepatitis C virus (HCV) genotype 1, body weight <85 kg, and high baseline viral load respond poorly to standard doses of pegylated interferon (peginterferon) and ribavirin. We evaluated intensified therapy with peginterferon alfa-2a plus ribavirin. Methods This double-blind randomized trial included HCV genotype 1-infected outpatients from hepatology clinics with body weight <85 kg and HCV RNA titer <400,000 IU/mL. Patients were randomized to 180 μg/wk peginterferon alfa-2a for 48 weeks plus 1200 mg/day ribavirin (standard of care) (group A, n = 191) or 1400/1600 mg/day ribavirin (group B, n = 189). Additional groups included 360 μg/wk peginterferon alfa-2a for 12 weeks then 180 μg/wk peginterferon alfa-2a for 36 weeks plus 1200 mg/day ribavirin (group C, n = 382) or 1400/1600 mg/day ribavirin (group D, n = 383). Follow-up lasted 24 weeks after treatment. Results Sustained virologic response rates (HCV RNA level <15 IU/mL at end of follow-up) in groups A, B, C, and D were 38%, 43%, 44%, and 41%, respectively. There were no significant differences among the 4 groups or between pooled peginterferon alfa-2a regimens (A + B vs C + D: odds ratio [OR], 1.08; 95% confidence interval [CI], 0.831.39; P = .584) or pooled ribavirin regimens (A + C vs B + D: OR, 1.00; 95% CI, 0.791.28; P = .974). Conclusions In patients infected with HCV genotype 1 who are difficult to treat (high viral load, body weight <85 kg), a 12-week induction regimen of peginterferon alfa-2a and/or higher-dose ribavirin is not more effective than the standard regimen. © 2010 AGA Institute.

Evolução de pacientes com AIDS pós cART: Evolução clínica e laboratorial de pacientes com AIDS após 48 semanas de tratamento antirretroviral

Combination Antiretroviral Therapy (cART) aims to inhibit viral replication, delay immunodeficiency progression and improve survival in AIDS patients. The objective of this study was to compare two different schemes of cART, based on plasma viral load (VL) and CD4+ T lymphocyte count, during 48 weeks of treatment. For this purpose, 472 medical charts of a Specialized Outpatient Service were reviewed from 1998 to 2005. Out of these, 58 AIDS patients who had received a triple drug scheme as the initial treatment were included in the study and two groups were formed: Group 1 (G1): 47 individuals treated with two nucleoside reverse-transcriptase inhibitors (NRTI) and one non-nucleoside reverse-transcriptase inhibitor; Group 2 (G2): 11 patients treated with two NRTI and one protease inhibitor. In G1 and G2, 53.2% and 81.8% respectively were patients with an AIDS-defining disease. The T CD4+ lymphocyte count increased progressively up until the 24th week of treatment in all patients, while VL became undetectable in 68.1% of G1 and in 63.6% of G2. The study concluded that the evolutions of laboratory tests were similar in the two treatment groups and that both presented a favorable clinical evolution.

Qualidade de vida e morbidade psicológica de pacientes portadores de hepatite C em tratamento com interferon peguilado e ribavirina

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)