Steroidal saponins from the roots of Solanum sisymbriifolium Lam. (Solanaceae) have inhibitory activity against dengue virus and yellow fever virus

Dengue is the most important arthropod-borne viral disease worldwide. Infection with any of the four dengue virus (DENV) serotypes can be asymptomatic or lead to disease with clinical symptoms ranging from undifferentiated and self-limiting fever to severe dengue disease, which can be fatal in some cases. Currently, no specific antiviral compound is available for treating DENV. The aim of this study was to identify compounds in plants from Paraguayan folk medicine with inhibitory effects against DENV. We found high virucidal activity (50% maximal effective concentration (EC50) value of 24.97 µg/mL) against DENV-2 in the ethanolic extract of the roots of Solanum sisymbriifolium Lam. (Solanaceae) without an evident cytotoxic effect on Vero E6 cells. Three saponins isolated from the root extract showed virucidal effects (EC50 values ranging from 24.9 to 35.1 µg/mL) against DENV-2. Additionally, the saponins showed inhibitory activity against yellow fever virus (EC50 values ranging from 126 to 302.6 µg/mL), the prototype virus of the Flavivirus genus, suggesting that they may also be effective against other members of this genus. Consequently, these saponins may be lead compounds for the development of antiviral agents.

Detección del virus influenza pandémica A (H1N1) en Paraguay 2009, y amplificación de genes hemaglutinina y neuraminidasa / Detection of pandemic influenza A (H1N1) virus in Paraguay in 2009, and amplification of the hemagglutinin and neuraminidase genes

Introducción: El virus de influenza pandémica A (H1N1), cuya circulación se inició en abril del año 2009 en México y Estados Unidos, se constituyó en el último virus pandémico desde los casos detectados en Hong Kong en 1968. El genoma del virus de influenza A está formado por 8 segmentos ARN de cadena simple (polaridad negativa), que codifican para 10 proteínas. Los genes hemaglutinina y neuraminidasa codifican para dos proteínas de superficie y son los utilizados en los análisis de variabilidad genética. Objetivos: a) Detectar la circulación del virus pandémico en pacientes con sospecha clínica de infección por influenza, y b) Diseñar una estrategia para amplificar de forma completa los genes hemaglutinina y neuraminidasa. Materiales y Métodos: Fueron analizados por Real-Time RT-PCR (transcripción reversa y reacción en cadena de la polimerasa en tiempo real) un total de 181 muestras de hisopado faríngeo, colectadas o remitidas al Hospital de Clínicas, del 6 de agosto al 11 de octubre de 2009. Para el diseño de amplificación de los genes hemaglutinina y neuraminidasa, se han utilizado herramientas bioinformáticas y reacción en cadena de la polimerasa. Resultados: Del total de muestras analizadas, 27 (14.9 %) dieron resultado positivo para el nuevo virus pandémico. Por otra parte, la amplificación completa de ambos genes proporcionó los resultados esperados: 1678-pares de bases (pb) para la hemaglutinina, y 1427-pb para la neuraminidasa. Conclusiones: La implementación de esta tecnología de amplificación permitirá posteriormente la secuenciación de estos genes a fin de determinar las variaciones genéticas del virus que podrían tener un impacto en la salud humana.

Introduction: The pandemic influenza A (H1N1) virus, whose circulation was detected in April 2009 in Mexico and the United States, is the latest pandemic virus since the cases reported in Hong Kong in 1968. The genome of the influenza A virus consists of 8 segments of single-stranded RNA of negative polarity, coding for 10 proteins. The hemagglutinin and neuraminidase genes encode for two surface proteins and are used in the analysis of genetic variability. Objectives: a) to detect circulation of the pandemic virus in patients with clinical suspicion of influenza infection and b) design a strategy to fully amplify the hemagglutinin and neuraminidase genes.Materials and Methods: A total of 181 pharyngeal swabs were collected and sent to the Hospital de Clínicas for analysis using Real-Time RT-PCR (reverse transcription and polymerase chain reaction in real time) between 6 August and 11 October 2009. To design the amplification of hemagglutinin and neuraminidase genes, we used bioinformatic tools and polimerase chain reaction. Results: Of the samples analyzed, 27 (14.9%) were positive for the new pandemic virus. Moreover, the complete amplification of both genes provided the expected results: 1678-base pairs (bp) for the hemagglutinin, and 1427-bp for neuraminidase. Conclusions: The use of this technology for amplification will eventually allow sequencing to identify genetic variations of the virus that could have an impact on human health.

Estudio de Fiebre Amarilla en primates en áreas de brote de los departamentos de San Pedro y Central del Paraguay / Study of Yellow Fever in primates in outbreaks areas of the departments of San Pedro and Central in Paraguay

La Fiebre Amarilla (FA) es una de las más importantes zoonosis que afecta a poblaciones humanas. La FA silvestre es imposible de ser erradicada, manteniéndose activa en zonas tropicales en África y Sudamérica. Todas las especies de primates son susceptibles y se consideran reservorios en el medio silvestre. La mortalidad es baja, se desconoce su valor con precisión, sin embargo existen epizootias con alta mortalidad, en humanos varía entre 20-50%. El objetivo de este trabajo fue buscar evidencias de FA en primates capturados en áreas de brote de FA de los departamentos de San Pedro y Central del Paraguay mediante la técnica de Neutralización por reducción de placas para FA cepa vacunal 17 D. Los resultados en los 35 primates estudiados fueron negativos, quizás por lo tardío del momento en la toma de muestras y bajo número de primates capturados.

Yellow Fever (YF) is one of the most important zoonotic diseases affecting human population. It is impossible to eradicate wild YF remaining active in tropical zones of Africa and South America. All species of primates are susceptible and are considered reservoirs in wild regions. Mortality is low and its precise value is unknown though there are epizootics with high mortality rates and in humans varies between 20-50%. The objective of this study was to search for evidence of YF in primates caught in YF outbreaks areas of the departments of San Pedro and Central in Paraguay through the neutralization technique by plates reduction for YF vaccine strain 17 D. The results in the 35 primates studied were negative, perhaps because of the lateness of the time sampling and the low number of captured primates.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Cytomegalovirus infection in renal transplant recipients diagnosed by nested-PCR

A prospective study of cytomegalovirus (CMV) infection was carried out on 34 renal transplant recipients managed at a General Hospital in Ribeirão Preto, SP, Brazil. Serologic tests showed that all patients were infected with CMV before renal transplantation. Two nested-PCR techniques with primers that recognize sequences of the glycoprotein B (gB) and H (gH) genes were used for CMV detection in blood and urine samples during the post-transplantation period. CMV was detected more frequently in blood samples than in urine samples (P<0.001). Thirty-three patients had CMV detected at least once in blood and/or urine samples. Seven of these patients (21.2 percent) were diagnosed as having symptomatic CMV infection and showed a worse clinical outcome, with a higher death rate (P = 0.03). No association between CMV viremia and graft rejection was observed. Nested-PCR was not useful to identify patients at risk for symptomatic CMV infection since only 21.2 percent of the patients with CMV infection were symptomatic

Diagnóstico de infecção congênita e perinatal por citomegalovírus utilizando a reação em cadeia da polimerase / Diagnosis of congenital and perinatal infection by cytomegalovirus using polymerase chain reaction

Aplicou-se uma reação em cadeia da polimerase (PCR) no diagnóstico de infecção congênita e perinatal por citomegalovirus, comparando-a com a técnica de isolamento viral em cultura celular. Foram processadas 305 amostras de urina de crianças de 0 a 6 meses, por ambas as técnicas. Utilizou-se na PCR os primers que amplificam parte do gene codificador do principal antígeno precoce imediato de CMV. Detectou-se virúria em 47 amostras por PCR e comparando os resultados com aqueles obtidos pelo isolamento viral, observou-se copositividade de 89,6% e conegatividade de 98,5%. Estas amostras positivas tiveram o resultado confirmado por PCR utilizando outros primers que amplificam regiões dos genes codificadores das glicoproteínas B e H de CMV. O diagnóstico de infecção congênita e perinatal por CMV pela PCR mostrou sensibilidade comparável à do isolamento viral e o uso de vários primers conferiu alta especificidade ao teste.

The practical application of a polymerase chain reaction (PCR) amplification for the diagnosis of congenital and perinatal cytomegalovirus (CMV) infections was evaluated. Three hundred five urine samples were tested by PCR and conventional virus isolation in cell culture. Viruria was detected in 47 urine samples by PCR using a primer pair which amplifies part of the major immediate-early (MIE) CMV genome. The PCR compared to virus isolation showed 89.6% sensitivity, 98.5% specificity and 91.5% positive predictive value. PCR with primer pairs amplifying parts of the glycoprotein B and glycoprotein H genes of CMV were used for confirmation of the positivity of the 47 urine samples. We concluded that this CMV PCR assay in urine has a suitable sensitivity for the diagnosis of congenital and perinatal infections and its specificity is highly increased by use of more than one pair of primers among the ones we used.