A simple one-step real-time RT-PCR for diagnosis of dengue virus infection.

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.

Ilheus and Saint Louis encephalitis viruses elicit cross-protection against a lethal Rocio virus challenge in mice.

Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.

Phospholipase A2 isolated from the venom of Crotalus durissus terrificus inactivates dengue virus and other enveloped viruses by disrupting the viral envelope.

The Flaviviridae family includes several virus pathogens associated with human diseases worldwide. Within this family, Dengue virus is the most serious threat to public health, especially in tropical and sub-tropical regions of the world. Currently, there are no vaccines or specific antiviral drugs against Dengue virus or against most of the viruses of this family. Therefore, the development of vaccines and the discovery of therapeutic compounds against the medically most important flaviviruses remain a global public health priority. We previously showed that phospholipase A2 isolated from the venom of Crotalus durissus terrificus was able to inhibit Dengue virus and Yellow fever virus infection in Vero cells. Here, we present evidence that phospholipase A2 has a direct effect on Dengue virus particles, inducing a partial exposure of genomic RNA, which strongly suggests inhibition via the cleavage of glycerophospholipids at the virus lipid bilayer envelope. This cleavage might induce a disruption of the lipid bilayer that causes a destabilization of the E proteins on the virus surface, resulting in inactivation. We show by computational analysis that phospholipase A2 might gain access to the Dengue virus lipid bilayer through the pores found on each of the twenty 3-fold vertices of the E protein shell on the virus surface. In addition, phospholipase A2 is able to inactivate other enveloped viruses, highlighting its potential as a natural product lead for developing broad-spectrum antiviral drugs.

Galectin-1 exerts inhibitory effects during DENV-1 infection.

Dengue virus (DENV) is an enveloped RNA virus that is mosquito-transmitted and can infect a variety of immune and non-immune cells. Response to infection ranges from asymptomatic disease to a severe disorder known as dengue hemorrhagic fever. Despite efforts to control the disease, there are no effective treatments or vaccines. In our search for new antiviral compounds to combat infection by dengue virus type 1 (DENV-1), we investigated the role of galectin-1, a widely-expressed mammalian lectin with functions in cell-pathogen interactions and immunoregulatory properties. We found that DENV-1 infection of cells in vitro exhibited caused decreased expression of Gal-1 in several different human cell lines, suggesting that loss of Gal-1 is associated with virus production. In test of this hypothesis we found that exogenous addition of human recombinant Gal-1 (hrGal-1) inhibits the virus production in the three different cell types. This inhibitory effect was dependent on hrGal-1 dimerization and required its carbohydrate recognition domain. Importantly, the inhibition was specific for hrGal-1, since no effect was observed using recombinant human galectin-3. Interestingly, we found that hrGal-1 directly binds to dengue virus and acts, at least in part, during the early stages of DENV-1 infection, by inhibiting viral adsorption and its internalization to target cells. To test the in vivo role of Gal-1 in DENV infection, Gal-1-deficient-mice were used to demonstrate that the expression of endogenous Galectin-1 contributes to resistance of macrophages to in vitro-infection with DENV-1 and it is also important to physiological susceptibility of mice to in vivo infection with DENV-1. These results provide novel insights into the functions of Gal-1 in resistance to DENV infection and suggest that Gal-1 should be explored as a potential antiviral compound.

Crotoxin and phospholipases A2 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 A2 showed a high inhibition of Yellow fever and dengue viruses in VERO E6 cells.

Comparação de testes nao treponêmicos utilizados no diagnóstico da sifilis e interferência da inativação do soro / Comparison of non treponemic assays used in syphilis diagnosis and interference of serum inactivation

A sífilis é uma doença infecciosa crônica causada pelo Treponema pallidum, cujo diagnóstico incorreto propicia a transmissão da doença via sexual e placentária. O trabalho avaliou comparativamente os testes Veneral Diseases Research Laboratory (VDRL) e Rapid Plasm Reagin (RPR) na triagem sorológica e determinou a interferência da inativação do soro. Foram empregados reagentes comerciais na avaliação de amostras de soro de 110 indivíduos saudáveis e de 20 pacientes portadores de sífilis, antes e após a inativação a 56ºC/30min. O procedimento técnico seguindo as instruções do fabricante mostrou sensibilidade de 100% para ambos os testes, embora 45% das amostras reativas tenham mostrado títulos superiores no VDRL. A especificidade foi de 100% no VDRL e 97,3% no RPR. A inativação do soro mostrou não interferir nos resultados do VDRL, enquanto que o RPR mostrou maior especificidade para amostras inativadas, embora sem diferença significativa (p=0,248).

Syphilis is a chronic infectious illness caused by the Treponema pallidum, which incorrect diagnosis propitiates the transmission mainly by sexual contact and from mother to child (trans-placentary). The present work evaluated the Veneral Diseases Research Laboratory (VDRL) and the Rapid Plasm Reagin (RPR) assays and also determined the interference of the serum inactivation. Commercial reagents were used to evaluate sera samples from 110 healthful individuals and 20 patients with syphilis, before and after inactivation at 56ºC/30min. In the technical procedure according the manufacturers’ instructions, both assays had presented 100% of sensitivity, although 45% of the reactive samples have shown bigger values in VDRL. The specificity was 100% for VDRL and 97.3% for RPR. The serum inactivation showed no interference in VDRL results, while in RPR was observed a greater specificity with inactivated samples, although with no significant difference (p=0,248).