CCR2 Plays a Protective Role in Rocio Virus-Induced Encephalitis by Promoting Macrophage Infiltration Into the Brain.

Rocio virus (ROCV) is a highly neuropathogenic mosquito-transmitted flavivirus responsible for an unprecedented outbreak of human encephalitis during 1975-1976 in Sao Paulo State, Brazil. Previous studies have shown an increased number of inflammatory macrophages in the central nervous system (CNS) of ROCV-infected mice, implying a role for macrophages in the pathogenesis of ROCV. Here, we show that ROCV infection results in increased expression of CCL2 in the blood and in infiltration of macrophages into the brain. Moreover, we show, using CCR2 knockout mice, that CCR2 expression is essential for macrophage infiltration in the brain during ROCV infection and that the lack of CCR2 results in increased disease severity and mortality. Thus, our findings show the protective role of CCR2-mediated infiltration of macrophages in the brain during ROCV infection.

Full genome sequence of Rocio virus reveal substantial variations from the prototype Rocio virus SPH 34675 sequence.

Rocio virus (ROCV) is an arbovirus belonging to the genus Flavivirus, family Flaviviridae. We present an updated sequence of ROCV strain SPH 34675 (GenBank: AY632542.4), the only available full genome sequence prior to this study. Using next-generation sequencing of the entire genome, we reveal substantial sequence variation from the prototype sequence, with 30 nucleotide differences amounting to 14 amino acid changes, as well as significant changes to predicted 3'UTR RNA structures. Our results present an updated and corrected sequence of a potential emerging human-virulent flavivirus uniquely indigenous to Brazil (GenBank: MF461639).

Chimeric viruses between Rocio and West Nile: the role for Rocio prM-E proteins in virulence and inhibition of interferon-α/ß signaling.

Mosquito-transmitted flavivirus Rocio (ROCV) was responsible for an outbreak of encephalitis in the Ribeira Valley, located in the south coast of Sao Paulo State, Brazil, in 1975-1976. ROCV also causes fatal encephalitis in adult mice. Seroprevalence studies in humans, horses and water buffaloes in different regions of Brazil have suggested that ROCV is still circulating in the country, indicating the risk of re-emergence of this virus. West Nile virus (WNV) is also a mosquito-transmitted encephalitic flavivirus, however, WNV strains circulating in Australia have not been associated with outbreaks of disease in humans and exhibit low virulence in adult mice. To identify viral determinants of ROCV virulence, we have generated reciprocal chimeric viruses between ROCV and the Australian strain of WNV by swapping structural prM and E genes. Chimeric WNV containing ROCV prM-E genes replicated more efficiently than WNV or chimeric ROCV containing WNV prM-E genes in mammalian cells, was as virulent as ROCV in adult mice, and inhibited type I IFN signaling as efficiently as ROCV. The results show that ROCV prM and E proteins are major virulence determinants and identify unexpected function of these proteins in inhibition of type I interferon response.

Influenza A H1N1pdm 2009 Virus in Paraguay: Nucleotide Point Mutations in Hemagglutinin and Neuraminidase Genes are not Associated with Drug Resistance.

Influenza virus is associated with upper respiratory tract infections. The fourth influenza pandemic was declared in 2009. The aim of this study was to determine the genetic variability of the 2009 H1N1 pandemic virus circulating in Paraguay. Nasal swabs were collected from 181 patients with flu symptoms managed at the Hospital of the Medical School in Asunción, Paraguay, between August and October 2009. Virus detection was carried out by real-time reverse transcription-polymerase chain reaction, followed by sequencing of the hemagglutinin and neuraminidase genes, and phylogenetic analysis. H1N1pdm09 was detected in 14.9% (27/181) of the suspected cases. Analysis of 13 samples showed that these viruses the clustered in a single genetic group. Neither the mutation related to exacerbation of disease (D239G in hemagglutinin) nor that related to antiviral resistance (H275Y in neuraminidase), both detected in neighboring countries, were found. This genetic analysis of H1N1pdm09 will help to understand the spread of the disease.

Mosquitoes infected with dengue viruses in Brazil.

Dengue epidemics have been reported in Brazil since 1985. The scenery has worsened in the last decade because several serotypes are circulating and producing a hyper-endemic situation, with an increase of DHF/DSS cases as well as the number of fatalities. Herein, we report dengue virus surveillance in mosquitoes using a Flavivirus genus-specific RT-Hemi-Nested-PCR assay. The mosquitoes (Culicidae, n = 1700) collected in the Northeast, Southeast and South of Brazil, between 1999 and 2005, were grouped into 154 pools. Putative genomes of DENV-1, -2 and -3 were detected in 6 mosquito pools (3.8%). One amplicon of putative DENV-1 was detected in a pool of Haemagogus leucocelaenus suggesting that this virus could be involved in a sylvatic cycle. DENV-3 was found infecting 3 pools of larvae of Aedes albopictus and the nucleotide sequence of one of these viruses was identified as DENV-3 of genotype III, phylogenetically related to other DENV-3 isolated in Brazil. This is the first report of a nucleotide sequence of DENV-3 from larvae of Aedes albopictus.

Predominance of rotavirus G2P[4] and emergence of G12P[9] strains in Asunción, Paraguay, 2006-2007.

Rotavirus is the most common cause of severe diarrhea in children worldwide. Monitoring the diversity of rotavirus strains is of great importance for current and future vaccination programs. To determine the diversity of rotavirus circulating in Asuncion, Paraguay, between 2006 and 2007, we carried out a molecular characterization of rotaviruses detected in children <5 years old and adults (>18 years old). We found that the most common circulating strain was G2P[4] (69/143), followed by G9P[8] (37/143). The temporal distribution of strains showed that, in children, G2P[4] was predominant in 2006, and that G2P[4] and G9P[8] were co-predominant in 2007, whereas in adults, G2P[4] was predominant in both years. Additionally, one G9P[6] and three G12P[9] strains were found in adult samples, making this the first report of these strains circulating in Paraguay. Sequence analysis of the G12P[9] strains suggests across-border migration of this strain within the southern cone of America.

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.

Genetic diversity of the E protein of dengue type 3 virus.

BACKGROUND: Dengue is the most important arbovirus disease in tropical and subtropical countries. The viral envelope (E) protein is responsible for cell receptor binding and is the main target of neutralizing antibodies. The aim of this study was to analyze the diversity of the E protein gene of DENV-3. E protein gene sequences of 20 new viruses isolated in Ribeirao Preto, Brazil, and 427 sequences retrieved from GenBank were aligned for diversity and phylogenetic analysis. RESULTS: Comparison of the E protein gene sequences revealed the presence of 47 variable sites distributed in the protein; most of those amino acids changes are located on the viral surface. The phylogenetic analysis showed the distribution of DENV-3 in four genotypes. Genotypes I, II and III revealed internal groups that we have called lineages and sub-lineages. All amino acids that characterize a group (genotype, lineage, or sub-lineage) are located in the 47 variable sites of the E protein. CONCLUSION: Our results provide information about the most frequent amino acid changes and diversity of the E protein of DENV-3.

Antiviral and antiparasite properties of an L-amino acid oxidase from the snake Bothrops jararaca: cloning and identification of a complete cDNA sequence.

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding alpha-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H2O2 production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pI of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs.

Molecular epidemiology of dengue type 3 virus in Brazil and Paraguay, 2002-2004.

We studied the molecular epidemiology of dengue virus type 3 (DENV-3) in Brazil and Paraguay by analyzing the 5' and 3' untranslated regions (5' and 3'UTRs) and the E protein gene of viruses isolated between 2002 and 2004. Both 5' and 3'UTRs were highly conserved. However, the 3'UTR of two isolates from Brazil contained eight nucleotide deletions compared with the remaining 26 viruses. Phylogenetic analyses suggested that DENV-3 was introduced into Brazil from the Caribbean Islands at least twice and into Paraguay from Brazil at least three times.

Hantavirus cardiopulmonary syndrome: immune response and pathogenesis.

Hantaviruses are emerging viruses in the Americas that cause cardiopulmonary syndrome with high lethality. The intense cellular immune response to hantavirus alters normal endothelial cell barrier functions and seems to be harmful to the host. On the other hand, the humoral immune response seems to be essential for recovery from infection.

Desenvolvimento de uma metodologia de PCR semiquantitativa utilizando plasmídio clonado com parte do gene gB de citomegalovírus / Development of a semi-quantitative PCR method using a plasmid cloned with part of the gB gene of cytomegalovirus

A infecçäo por citomegalovírus é disseminada em nosso meio e costuma acometer, com significante morbimortalidade, indivíduos imunodeprimidos, especialmente, transplantados de medula óssea e de rim bem como pacientes com AIDS. Neste trabalho, descrevemos o desenvolvimento de uma PCR semiquantitativa para detectar e quantificar cargas de CMV, presentes em materiais clínicos. Para tanto, inserimos em plasmídios PCR II (Invitrogen, USA), o fragmento com 296 pares-base do gene da glicoproteína B do CMV. Os plasmídios com inserto foram transfectados em Escherichia coli, multiplicados, verificados quanto à presença do inserto por seqüenciamento nucleotídico, purificados, e quantificados. Os plasmídios com inserto foram titulados em diluições decimais e as mesmas foram submetidas à PCR descrita anteriormente, que foi, também, utilizada nos testes semiquantitativos, permitindo determinar a sensibilidade da técnica, 867 cópias de CMV / mg de DNA. Com base nas densidades das bandas eletroforéticas dos amplicons de amostras clínicas, comparadas às da titulaçäo de plasmídios contendo inserto de glicoproteína B do CMV, obtivemos as cargas virais. A técnica de PCR semiquantitativa, por nós padronizada, tem, como vantagens, o baixo custo e o fácil manuseio após sua padronizaçäo, podendo ser testada na detecçäo da carga de CMV em diferentes tipos de pacientes com suspeita de citomegalovirose