Human rotavirus strains circulating in Venezuela after vaccine introduction: predominance of G2P[4] and reemergence of G1P[8].

BACKGROUND: Rotavirus (RV) is the most common cause of severe childhood diarrhea worldwide. Despite Venezuela was among the first developing countries to introduce RV vaccines into their national immunization schedules, RV is still contributing to the burden of diarrhea. Concerns exist about the selective pressure that RV vaccines could exert on the predominant types and/or emergence of new strains. RESULTS: To assess the impact of RV vaccines on the genotype distribution 1 year after the vaccination was implemented, a total of 912 fecal specimens, collected from children with acute gastroenteritis in Caracas from February 2007 to April 2008, were screened, of which 169 (18.5%) were confirmed to be RV positive by PAGE. Rotavirus-associated diarrhea occurred all year-round, although prevailed during the coolest and driest months among unvaccinated children under 24 months old. Of 165 RV strains genotyped for G (VP7) and P (VP4) by seminested multiplex RT-PCR, 77 (46.7%) were G2P[4] and 63 (38.2%) G1P[8]. G9P[8], G3P[8] and G2P[6] were found in a lower proportion (7.3%). Remarkable was also the detection of <5% of uncommon combinations (G8P[14], G8P[4], G1P[4] and G4P[4]) and 3.6% of mixed infections. A changing pattern of G/P-type distribution was observed during the season studied, with complete predominance of G2P[4] from February to June 2007 followed by its gradual decline and the reemergence of G1P[8], predominant since January 2008. Phylogenetic analysis of VP7 and VP4 genes revealed a high similarity among G2P[4] and global strains belonging to G2-II and P[4]-V lineages. The amino acid substitution 96D → N, related with reemergence of the G2 genotype elsewhere, was observed. The G1P[8] strains from Caracas were grouped into the lineages G1-I and P[8]-III, along with geographically remote G1P[8] rotaviruses, but they were rather distant from Rotarix® vaccine and pre-vaccine strains. Unique amino acid substitutions observed on neutralization domains of the VP7 sequence from Venezuelan post-vaccine G1P[8] could have conditioned their re-emergence and a more efficient dissemination into susceptible population. CONCLUSIONS: The results suggest that natural fluctuations of genotypes in combination with forces driving the genetic evolution could determine the spread of novel strains, whose long-term effect on the efficacy of available vaccines should be determined.

Circulación de virus Chikungunya en el estado Aragua (Venezuela) durante el año 2014 / Circulation of Chikungunya virus in Aragua state (Venezuela) during the year 2014

El virus chikungunya (CHIKV) es un Alfavirus causante de la fiebre chikungunya (CHIKF). En Venezuela, una región desprovista de inmunidad contra CHIKV y con presencia de Aedes aegypti y Aedes albopictus, el primer caso importado fue reportado por las autoridades sanitarias en junio de 2014. Por la relevancia del hecho, se analizaron 94 muestras de pacientes febriles que acudieron a los centros de salud públicos y privados del estado Aragua entre enero y diciembre de 2014, mediante la detección de los fragmentos de los genes nsP4 (Alfavirus) y E1 (CHIKV) utilizando técnicas moleculares, como Transcripción Reversa acoplada a Reacción en Cadena de la Polimerasa (RT-PCR) y/o secuenciación nucleotídica. Los resultados indicaron positividad en 19,2 % de las muestras analizadas. Se vieron afectados pacientes con edades entre 6 y 66 años, con predominio del sexo femenino (12/18). Clínicamente, todos los pacientes positivos a CHIKV manifestaron signos y síntomas asociados a CHIKF, tales como fiebre (18/18), artralgia (18/18) y erupción (16/18), entre otros. A pesar de que la positividad puede considerarse baja con relación a lo reportado en otras comunidades, este estudio representa el primer reporte local de detección molecular de CHIKV en Venezuela (estado Aragua) durante el año 2014.

Chikungunya virus is an Alphavirus that causes chikungunya Fever (CHIKF). In Venezuela, a region devoid of immunity against CHIKV and presence of Aedes aegypti and Aedes albopictus. The first imported case was reported by health authorities in June 2014. The relevance of the fact, 94 samples of febrile patients who came to the centers of public and private health Aragua state between january and december for detection of the nsP4 (Alphavirus) and E1 (CHIKV) fragments were analyzed by molecular techniques (Reverse Transcriptase Polymerase Chain Reaction and/or nucleotide sequencing). The results showed 19.2 % of positivity by CHIKV. Clinically all CHIKV positive patients showed signs and symptoms related with CHIKF, such as fever (18/18), arthralgia (18/18) and rash (16/18), among others. Were affected patients between the ages of 6 and 66 years with a predominance of the female sex (12/18). Although the positivity may be considered low compared to those reported in other communities, this represents the first local report of molecular detection of CHIKV in Venezuela (Aragua state) during 2014.

[Selection of heparin-sensitive dengue virus variants in BHK-21 cells]. / Selección de variantes de virus dengue sensibles a la heparina en células BHK-21.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.

Selección de variantes de virus dengue sensibles a la heparina en células BHK-21 / Selection of heparin-sensitive dengue virus variants in BHK-21 cells

Estudios previos han demostrado que la adaptación de diversos virus a crecer en líneas celulares de vertebrados, conduce a la selección de variantes virales que unen al heparán sulfato (HS) con alta afinidad. En el presente trabajo se determinó la susceptibilidad de cepas del virus dengue (DENV) a la heparina hipersulfatada un análogo al HS, después de pases seriados en células BHK-21. A aislados de campo de los cuatro serotipos de DENV, se les realizaron ocho pases seriados en células BHK-21. La adaptación de los DENV al cultivo celular seleccionó variantes virales con una aumentada capacidad replicativa en células BHK-21 y una incrementada susceptibilidad a la heparina, en relación a las respectivas cepas no adaptadas, obteniéndose una inhibición de la infectividad más significativa en DENV-2 y DENV-4. Las cepas de DENV adaptadas presentaron cambios en la secuencia de aminoácidos de la proteína de envoltura (E), en particular una substitución K204R para DENV-1, N67K para DENV-2, K308R y V452A para DENV-3 y E327G para DENV-4. Estas sustituciones implicaron ganancia de residuos básicos que incrementaron la carga neta positiva de la proteína. Los resultados sugieren, que la adaptación de cepas de DENV a células BHK-21 selecciona variantes virales sensibles a la heparina y que la efectividad de este compuesto varía dependiendo de la cepa viral. Además sugieren que el HS puede jugar un papel importante en la infectividad de las cepas de DENV adaptadas al cultivo celular, a diferencia de los aislados de DENV no adaptados.

Several studies have shown that adaptation of various viruses to grow in certain cell lines of vertebrates, leads to the selection of virus variants that bind heparan sulfate (HS) with high affinity. In this study we investigated the susceptibility of strains of dengue virus (DENV) to oversulfated heparin an analogue of HS after passages in BHK-21 cells. Field isolates of the four serotypes of DENV with a limited number of passes in mosquito cells C6/36HT were serially passaged eight times in BHK-21 cells. The adaptation of the DENV to the cell culture selected viral variants with an increased replicative capacity in BHK-21 cells and an increased susceptibility to heparin compared with the original not adapted strains, with a more significant inhibition of the infectivity in DENV-2 and DENV-4.The E protein of the adapted strains showed changes in the amino acid sequence, particularly at the position K204R to DENV-1, N67K to DENV-2, K308R and V452A for DENV-3 and E327G to DENV-4. These substitutions implicated a gain of basic residues that increased the net positive charge of the protein. These results suggest that adaptation of DENV strains to BHK-21 cells implies changes in the envelope protein, changes associated to the protein reactivity with heparin, the inhibitory effectiveness of this compound varying depending on the viral strain. In addition, these results suggest that the HS can play an important role in the infectivity of the DENV strains adapted to vertebrate cell culture, but not in the infectivity of non-adapted DENV isolates.