Detection and diversity of gastrointestinal viruses in wastewater from Caracas, Venezuela, 2021-2022.

Gastrointestinal viruses (GIV) are an important cause of childhood morbidity and mortality, particularly in developing countries. Their epidemiological impact in Venezuela during the COVID-19 pandemic remains unclear. GIV can also be detected in domestic sewage. Ninety-one wastewater samples from urban areas of Caracas collected over 12 months and concentrated by polyethylene-glycol-precipitation, were analyzed by multiplex reverse-transcription-PCR for rotavirus/calicivirus/astrovirus and enterovirus/klassevirus/cosavirus, and monoplex-PCR for adenovirus and Aichi virus. The overall frequency of virus detection was 46.2%, fluctuating over months, and peaking in the rainy season. Adenoviruses circulated throughout the year, especially type F41, and predominated (52.7%) over caliciviruses (29.1%) that peaked in the rainy months, rotaviruses (9.1%), cosaviruses (5.5%), astroviruses and enteroviruses (1.8%). Aichi-virus and klassevirus were absent. Rotavirus G9/G12, and P[4]/P[8]/P[14] predominated. The occurrence of GIV in wastewater reflects transmission within the population of Caracas and the persistence of a potential public health risk that needs to be adequately monitored.

Molecular characterization of human adenovirus causing infantile acute gastroenteritis in Venezuela before and after rotavirus vaccine implementation.

Human adenoviruses (HAdV) of species F are commonly involved in pediatric acute gastroenteritis (AGE). The real impact on Venezuelan health is unknown. To investigate the prevalence and molecular diversity of HAdV in Venezuela, 630 fecal samples collected from children with AGE in 3 cities, from 2001 to 2013, were tested by PCR. Species F and types F40/41 were identified by REA. HAdV was detected in 123 cases (19.5%), most from outpatient females under 24 months old. A progressive and substantial increase in the detection rate was observed over time, significantly higher in rotavirus vaccinated than unvaccinated children (28.4% vs. 9.5%, P = 0.00019). Phylogenetic analysis of 28 randomly selected genomes showed high similarity among HAdV-F40/41 and those worldwide. HAdV-F of type 41 prevailed (79.8%) and clustered into 2 intratypic major clades. The significant involvement of HAdV-F41 in AGE suggests the importance of actively monitoring viral agents other than rotavirus, especially after vaccine introduction.

Sub-lineages of the Omicron variant of SARS-CoV-2: characteristic mutations and their relation to epidemiological behavior / Sub-linajes de la variante Ómicron del SARS-CoV-2: mutaciones características y su relación con el comportamiento epidemiológico

Abstract By the end of 2021, the Omicron variant of SARS-CoV-2, the coronavirus responsible for COVID-19, emerges, causing immediate concern, due to the explosive increase in cases in South Africa and a large number of mutations. This study describes the characteristic mutations of the Omicron variant in the Spike protein, and the behavior of the successive epidemic waves associated to the sub-lineages throughout the world. The mutations in the Spike protein described are related to the virus ability to evade the protection elicited by current vaccines, as well as with possible reduced susceptibility to host proteases for priming of the fusion process, and how this might be related to changes in tropism, a replication enhanced in nasal epithelial cells, and reduced in pulmonary tissue; traits probably associated with the apparent reduced severity of Omicron compared to other variants.

Resumen A finales de 2021 surge la variante Omicron del SARS-CoV-2, el coronavirus responsable de la COVID-19, causando preocupación inmediata, debido al aumento explosivo de casos en Suráfrica, y a su gran cantidad de mutaciones. Este estudio describe las mutaciones características de la variante Ómicron en la proteína de la Espiga (S) y el comportamiento de las sucesivas olas epidémicas asociadas a la circulación de sus sub-linajes en todo el mundo. Las mutaciones en la proteína S descritas están relacionadas con su capacidad para evadir la protección provocada por las vacunas actuales, así como su posible susceptibilidad reducida a las proteasas del hospedero para la preparación del proceso de fusión. Se infiere cómo esto podría estar relacionado con su cambio en el tropismo, con una replicación mayor en las células epiteliales nasales y menor en el tejido pulmonar, rasgos probablemente asociados a su aparente menor gravedad en comparación con otras variantes.

Introduction and rapid dissemination of SARS-CoV-2 Gamma Variant of Concern in Venezuela.

In less than two years since SARS-CoV-2 emerged, the new coronavirus responsible for COVID-19, has accumulated a great number of mutations. Many of these mutations are located in the Spike protein and some of them confer to the virus higher transmissibility or partial resistance to antibody mediated neutralization. Viral variants with such confirmed abilities are designated by WHO as Variants of Concern (VOCs). The aim of this study was to monitor the introduction of variants and VOCs in Venezuela. A small fragment of the viral genome was sequenced for the detection of the most relevant mutations found in VOCs. This approach allowed the detection of Gamma VOC. Its presence was confirmed by complete genome sequencing. The Gamma VOC was detected in Venezuela since January 2021, and in March 2021 was predominant in the East and Central side of the country, representing more than 95% of cases sequenced in all the country in April-May 2021. In addition to the Gamma VOC, other isolates carrying the mutation E484K were also detected. The frequency of this mutation has been increasing worldwide, as shown in a survey of sequences carrying E484K mutation in GISAID, and was detected in Venezuela in many probable cases of reinfection. Complete genome sequencing of these cases allowed us to identify E484K mutation in association with Gamma VOC and other lineages. In conclusion, the strategy adopted in this study is suitable for genomic surveillance of variants for countries lacking robust genome sequencing capacities. In the period studied, Gamma VOC seems to have rapidly become the dominant variant throughout the country.

Molecular detection of human enteric viruses circulating among children with acute gastroenteritis in Valencia, Venezuela, before rotavirus vaccine implementation.

BACKGROUND: The role of rotavirus as main etiologic agent of diarrhea has been well documented worldwide, including in Venezuela. However, information about the prevalence of gastrointestinal viruses such as calicivirus, adenovirus and astrovirus is limited and the contribution of other agents as Aichi virus and klassevirus is largely unknown. To explore the etiological spectrum of diarrhea associated with agents other than rotaviruses, 227 stool samples from children under 5 years old with acute gastroenteritis, collected in Valencia (Venezuela) from 2001 to 2005, and previously tested as rotavirus-negative, were analyzed for caliciviruses, adenoviruses, astroviruses, Aichi viruses, klasseviruses, picobirnaviruses and enteroviruses by specific RT-PCRs. RESULTS: At least one viral agent was detected in 134 (59%) of the samples analyzed, mainly from children under 24 months of age and most of them belonging to the lowest socioeconomic status. Overall, enterovirus was identified as the most common viral agent (37.9%), followed by calicivirus (23.3%), adenovirus (11.5%), astrovirus (3.5%), klassevirus (1.3%) and Aichi virus (0.4%), while no picobirnavirus was detected. Klasseviruses were found during 2004 and 2005 and Aichi viruses only in 2005, indicating their circulation in Venezuela; meanwhile, the rest of the viruses were detected during the whole study period. Coinfections with two or more viruses were found in 39 (29.1%) of the infected children, most under 24 months of age. Adenovirus was involved as the coinfecting agent in at least 46.9% of the cases, but no differences concerning socio-demographic variables were observed between the coinfected and the single infected children. CONCLUSIONS: The results show that various enteric viruses, including enteroviruses, caliciviruses and adenoviruses, accounted for a significant proportion of infantile diarrhea cases in Venezuela before rotavirus vaccine implementation. In addition, emerging viruses as Aichi virus and klassevirus were found, indicating the need to continue monitoring their spreading into the communities. Efforts are needed to develop more accurate methods to identify the major causes of diarrhea and to provide tools for more effective preventive measures.

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.

Prevalence and molecular characterization of G6PD deficiency in two Plasmodium vivax endemic areas in Venezuela: predominance of the African A-(202A/376G) variant.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency causes acute haemolytic anaemia triggered by oxidative drugs such as primaquine (PQ), used for Plasmodium vivax malaria radical cure. However, in many endemic areas of vivax malaria, patients are treated with PQ without any evaluation of their G6PD status. METHODS: G6PD deficiency and its genetic heterogeneity were evaluated in northeastern and southeastern areas from Venezuela, Cajigal (Sucre state) and Sifontes (Bolívar state) municipalities, respectively. Blood samples from 664 randomly recruited unrelated individuals were screened for G6PD activity by a quantitative method. Mutation analysis for exons 4-8 of G6PD gen was performed on DNA isolated from G6PD-deficient (G6PDd) subjects through PCR-RFLP and direct DNA sequencing. RESULTS: Quantitative biochemical characterization revealed that overall 24 (3.6%) subjects were G6PDd (average G6PD enzyme activity 4.5 ± 1.2 U/g Hb, moderately deficient, class III), while DNA analysis showed one or two mutated alleles in 19 of them (79.2%). The G6PD A-(202A/376G) variant was the only detected in 17 (70.8%) individuals, 13 of them hemizygous males and four heterozygous females. Two males carried only the 376A → G mutation. No other mutation was found in the analysed exons. CONCLUSIONS: The G6PDd prevalence was as low as that one shown by nearby countries. This study contributes to the knowledge of the genetic background of Venezuelan population, especially of those living in malaria-endemic areas. Despite the high degree of genetic mixing described for Venezuelan population, a net predominance of the mild African G6PD A-(202A/376G) variant was observed among G6PDd subjects, suggesting a significant flow of G6PD genes from Africa to Americas, almost certainly introduced through African and/or Spanish immigrants during and after the colonization. The data suggest that 1:27 individuals of the studied population could be G6PDd and therefore at risk of haemolysis under precipitating factors. Information about PQ effect on G6PDd individuals carrying mild variant is limited, but since the regimen of 45 mg weekly dose for prevention of malaria relapse does not seem to be causing clinically significant haemolysis in people having the G6PD A-variant, a reasoned weighing of risk-benefit for its use in Venezuela should be done, when implementing public health strategies of control and elimination.

Glucosa 6 fosfato deshidrogenasa: características bioquímicas y moleculares. Prevalencia de la deficiencia / Glucose-6-phosphate dehydrogenase: biochemical and molecular characteristics. Prevalence of deficiency

La glucosa-6-fosfato deshidrogenasa (G6PD) es una enzima citoplasmática que se encuentra distribuida en todas las células del organismo y que cataliza el primer paso de la vía de las pentosas en el cual la glucosa 6 fosfato (G6P) es convertida a 6-fosfogluconato(6FG) y el NADP reducido a NADPH, proceso indispensable para proteger a los eritrocitos del daño oxidativo. La deficiencia de la enzima G6PD es la eritroenzimopatía más común, es recesiva y ligada al cromosoma X, con amplia distribución mundial y elevada heterogeneidad genética y bioquímica. Se realizó una revisión sobre aspectos bioquímicos, estructura, genética, bases moleculares, defecto enzimático, prevalencia de la deficiencia en el mundo y en Venezuela, y el papel de la deficiencia de G6PD en el tratamiento de la malaria por el incremento en el riesgo de hemolisis que lleva consigo la tendencia de aumentar la dosis total de Primaquina en sujetos paludismo,particularmente en las regiones donde predomina la infección por Plasmodium vivax.

[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.

[Enteropathogens responsible for gastrointestinal disorders in HIV patients]. / Agentes patógenos responsables de trastornos gastrointestinales en sujetos infectados con VIH.

Gastrointestinal disorders or GID are debilitating conditions common in individuals infected by the human immunodeficiency virus (HIV), capable of leading to death. Numerous etiological agents and pathophysiological mechanisms have been involved in this status. Although the use of highly active antiretroviral therapy (HAART) in many countries has greatly reduced the prevalence of gastrointestinal infections, enteric pathogens such as bacteria, parasites, fungi and viruses may still act as opportunist agents in these patients. Cytomegalovirus, adenovirus, calicivirus, astrovirus, rotavirus, enterovirus, picobirnavirus and some more recently described, like bocavirus and Aichi virus, have been detected in HIV patients. However, except for cytomegalovirus, which is an established etiological agent of GID in these patients, the role of the other viruses remains unclear. Several species of Cryptosporidium, microsporidia, Salmonella, atipical mycobacteria and Campylobacter jejuni, have also been recognized as important causes of GID in HIV patients. The progressive incorporation of increasingly sensitive immunological and molecular assays for antigen, antibody and pathogens detection from faeces, has improved the diagnosis of diarrhea and contributed to clarify the etiological significance of some microorganisms in immunocompetent patients. In Venezuela, some information is available about the prevalence of enteric pathogens in immunocompromised patients infected with HIV. The identification of the etiologic agent responsible for this condition may be useful for the management and treatment of these patients, for whom viral enteritis is a disease, which reduces their quality of life and causes a high public health spending.

Agentes patógenos responsables de trastornos gastrointestinales en sujetos infectados con VIH / Enteropathogens responsible of gastrointestinal disorders in HIV patients

Los trastornos gastrointestinales o TGI son afecciones debilitantes muy comunes en individuos infectados con el virus de inmunodeficiencia humana (VIH), que pueden conducir a muerte. Numerosos agentes etiológicos y mecanismos patofisiológicos han sido propuestos causar esta afección. A pesar del uso de terapia antirretroviral, que ha reducido enormemente la prevalencia de TGI en estos pacientes, patógenos entéricos como virus, bacterias, parásitos y hongos logran actuar todavía como agentes oportunistas. Citomegalovirus, adenovirus, calicivirus, astrovirus, rotavirus, enterovirus, picobirnavirus y algunos más recientemente descritos, como bocavirus y Aichi virus han sido detectados en pacientes con VIH. Sin embargo, a excepción del citomegalovirus, hay muy poca certeza acerca del papel que juegan algunos de ellos en estas afecciones. Varias especies de Criptosporidium, microsporidos, Salmonella, micobacterias atípicas y Campylobacter jejuni han sido reconocidos también como una importante causa de TGI en estos pacientes. La progresiva incorporación de técnicas inmunoenzimáticas y moleculares, cada vez más sensibles para la detección de antígenos, anticuerpos y agentes patógenos en heces ha mejorado el diagnóstico de las diarreas y contribuido a esclarecer la importancia etiológica de algunos microorganismos en los pacientes inmunocompetentes. En Venezuela existen algunos datos acerca de la prevalencia de patógenos entéricos en pacientes inmunodeficientes infectados con VIH. La identificación del agente etiológico responsable de TGI podría ser de gran utilidad para el manejo y tratamiento de estos pacientes, para quienes la enteritis viral es una manifestación morbosa que reduce la calidad de vida y ocasiona un elevado gasto en salud pública.

Gastrointestinal disorders or GID are debilitating conditions common in individuals infected by the human immunodeficiency virus (HIV), capable of leading to death. Numerous etiological agents and pathophysiological mechanisms have been involved in this status. Although the use of highly active antiretroviral therapy (HAART) in many countries has greatly reduced the prevalence of gastrointestinal infections, enteric pathogens such as bacteria, parasites, fungi and viruses may still act as opportunist agents in these patients. Cytomegalovirus, adenovirus, calicivirus, astrovirus, rotavirus, enterovirus, picobirnavirus and some more recently described, like bocavirus and Aichi virus, have been detected in HIV patients. However, except for cytomegalovirus, which is an established etiological agent of GID in these patients, the role of the other viruses remains unclear. Several species of Cryptosporidium, microsporidia, Salmonella, atipical mycobacteria and Campylobacter jejuni, have also been recognized as important causes of GID in HIV patients. The progressive incorporation of increasingly sensitive immunological and molecular assays for antigen, antibody and pathogens detection from faeces, has improved the diagnosis of diarrhea and contributed to clarify the etiological significance of some microorganisms in immunocompetent patients. In Venezuela, some information is available about the prevalence of enteric pathogens in immunocompromised patients infected with HIV. The identification of the etiologic agent responsible for this condition may be useful for the management and treatment of these patients, for whom viral enteritis is a disease, which reduces their quality of life and causes a high public health spending.

Rotavirus infection of cells in culture induces activation of RhoA and changes in the actin and tubulin cytoskeleton.

Rotavirus infection induces an increase in [Ca(2+)](cyto), which in turn may affect the distribution of the cytoskeleton proteins in the infected cell. Changes in microfilaments, including the formation of stress fibers, were observed starting at 0.5 h.p.i. using fluorescent phalloidin. Western blot analysis indicated that RhoA is activated between 0.5 and 1 h.p.i. Neither the phosphorylation of RhoA nor the formation of stress fibers were observed in cells infected with virions pre-treated with an anti-VP5* non-neutralizing mAb, suggesting that RhoA activation is stimulated by the interaction of the virus with integrins forming the cell receptor complex. In addition, the structure of the tubulin cytoskeleton was also studied. Alterations of the microtubules were evident starting at 3 h.p.i. and by 7 h.p.i. when microtubules were markedly displaced toward the periphery of the cell cytoplasm. Loading of rotavirus-infected cells with either a Ca(2+) chelator (BAPTA) or transfection with siRNAs to silence NSP4, reversed the changes observed in both the microfilaments and microtubules distribution, but not the appearance of stress fibers. These results indicate that alterations in the distribution of actin microfilaments are initiated early during infection by the activation of RhoA, and that latter changes in the Ca(2+) homeostasis promoted by NSP4 during infection may be responsible for other alterations in the actin and tubulin cytoskeleton.

Genotyping of human rotaviruses circulating among children with diarrhea in Valencia, Venezuela.

Rotavirus infection is the most common cause of severe gastroenteritis during childhood worldwide, especially in developing countries. Two rotavirus vaccines are available for childhood immunization programs. Evaluation of the vaccine performance will benefit from knowledge of the epidemiological features of rotavirus infection in regional settings. Limited information on the molecular characteristics of the rotavirus types circulating in Venezuela is available. Eighty seven (89.7%) of the 97 ELISA rotavirus positive stool samples collected from children with diarrhea aged <5 years during 2003 in Valencia (Carabobo State), were G-, P- and NSP4-genotyped by RT-PCR and/or automated sequencing. Four common combinations, G3P[8]/NSP4-E1, G2P[4]/NSP4-E2, G9P[8]/NSP4-E1, and G1P[8]/NSP4-E1 were responsible for 50.6%, 35.6%, 5.7%, and 1.1%, respectively of cases of rotavirus diarrhea, most of them (66%) in children ≤12 months. One uncommon G8P[14]/NSP4-E2 strain was also detected. Temporal fluctuation of genotype distribution occurred, but no differences by age, diarrhea severity score, sex, treatment type or patient medical attention were observed, except for the G3P[8]/NSP4-E1, associated with a more severe dehydration than any other type (P < 0.01). The results confirm the broad diversity among rotavirus strains circulating in Venezuela prior to vaccine implementation, showing the predominance of G3, significant proportion of G2 and moderate circulation of G9 strains. Epidemiological surveillance is needed to detect the emergence of new genotypes that could escape protection induced by vaccination.

Seroepidemiology of porcine enteric sapovirus in pig farms in Venezuela.

Porcine enteric sapovirus (PES) has been shown to cause diarrhea under experimental conditions in gnotobiotic piglets. However, the role of PES as enteric pathogens in porcine farms remains unclear. To further understand the PES-host interactions under field conditions, a serological survey was carried out. To this end the capsid gene of a PES isolate was cloned in the baculovirus expression system and an ELISA was developed based on virus-like particles from the baculovirus-expressed PES capsid protein. A total of 85 serum samples collected from pigs ranging from 8 weeks to over 54 weeks of age were analyzed. An overall seroprevalence to PESs of 62% was found, with significant differences (p<0.05) found between ages. Pigs younger than 10 weeks old and older than 12 weeks old showed high seroprevalences (70-100%), while pigs aged 10-12 weeks showed no detectable serum antibodies levels. Our results suggest that PES infections are common in pigs and that passively acquired maternal antibodies are soon replaced by actively acquired antibodies, whose titers increase gradually with age and that probably are maintained during lifetime.

Molecular detection and characterization of Aichi viruses in sewage-polluted waters of Venezuela.

The circulation of Aichi virus in a major urban area was demonstrated using molecular detection with samples recovered from a major river polluted with sewage discharges in Caracas, Venezuela. Five out of 11 water samples studied were positive, being classified by phylogenetic analysis as genotype B. Analysis of sewage waters appears to be a useful methodology to uncover the presence of a hitherto undetected fecal pathogen in a given geographical area.

Silencing of rotavirus NSP4 or VP7 expression reduces alterations in Ca2+ homeostasis induced by infection of cultured cells.

Rotavirus infection of cells in culture induces major changes in Ca(2+) homeostasis. These changes include increases in plasma membrane Ca(2+) permeability, cytosolic Ca(2+) concentration, and total cell Ca(2+) content and a reduction in the amount of Ca(2+) released from intracellular pools sensitive to agonists. Various lines of evidence suggest that the nonstructural glycoprotein NSP4 and possibly the major outer capsid glycoprotein VP7 are responsible for these effects. In order to evaluate the functional roles of NSP4 and other rotavirus proteins in the changes in Ca(2+) homeostasis observed in infected cells, the expressions of NSP4, VP7, and VP4 were silenced using the short interfering RNA (siRNA) technique. The transfection of specific siRNAs resulted in a strong and specific reduction of the expression of NSP4, VP7, and VP4 and decreased the yield of new viral progeny by more than 90%. Using fura-2 loaded cells, we observed that knocking down the expression of NSP4 totally prevented the increase in Ca(2+) permeability of the plasma membrane and cytosolic Ca(2+) concentration measured in infected cells. A reduction in the levels of VP7 expression partially reduced the effect of infection on plasma membrane Ca(2+) permeability and Ca(2+) pools released by agonist (ATP). In addition, the increase of total Ca(2+) content (as measured by (45)Ca(2+) uptake) observed in infected cells was reduced to the levels in mock-infected cells when NSP4 and VP7 were silenced. Finally, when the expression of VP4 was silenced, none of the disturbances of Ca(2+) homeostasis caused by rotaviruses in infected cells were affected. These data altogether indicate that NSP4 is the main protein responsible for the changes in Ca(2+) homeostasis observed in rotavirus-infected cultured cells. Nevertheless, VP7 may contribute to these effects.

Estudio de la infección por rotavirus y calicivirus en neonatos de la Maternidad "Concepción Palacios" de Caracas / Study of rotavirus and calicivirus infections in neonates from the Maternity Hospital "Concepción Palacios", Caracas

En un estudio realizado en 1984 en la Maternidad "Concepción Palacios", Pérez-Schael y col. detectaron excreción asintomática de rotavirus en el 56% de los recién nacidos evaluados. El objetivo de este trabajo fue estudiar la infección por rotavirus en neonatos de la Maternidad y comparar datos con aquellos obtenidos anteriormente. Además, se investigó la presencia de calicivirus en dicha población. Entre agosto y diciembre de 2004, se recolectaron 307 muestras de heces provenientes de 215 neonatos sanos. Para la detección de los agentes virales se utilizaron ensayos tipo ELISA comerciales y no comerciales. Estos ensayos identificaron como positivas a rotavirus y calicivirus a un total de 14 y 58 muestras, respectivamente. Sin embargo, al realizar pruebas para corroborar la presencia de rotavirus por microscopia electrónica, EGPA y RT-PCR y para calicivirus por RT-PCR, ninguna de las muestras señaladas como positivas pudo ser confirmada. Estos resultados sugieren la no-circulación tanto de rotavirus como de calicivirus dentro de la Maternidad. Es posible que la interrupción de la transmisión de rotavirus dentro de la Institución pueda deberse a cambios en el manejo del par madre-neonato introducidos en la Maternidad desde 1995, promovidos por la OMS y UNICEF.

In a study carried out in 1984 at the "Concepción Palacios" Maternity Hospital, Perez-Schael et al (J Med Virol 1984, 14:127) detected asymptomatic excretion of rotavirus in 56% of the neonates evaluated. The purpose of this work was to study rotavirus infection in neonates at the Maternity Hospital and compare the new data with those previously obtained. We also studied calicivirus presence in said population. Between August and December 2004, 307 feces samples were collected from 215 healthy neonates. Commercial and non commercial ELISA type assays were used for detection of viral agents. These assays identified a total of 14 and 58 samples as rotavirus and calicivirus positive respectively. Nevertheless, when carrying out tests to corroborate rotavirus presence with electron microscopy, EGPA and RT-PCR, and for calicivirus with RT-PCR, none of the samples previously shown as positive could be confirmed. These results suggest the non-circulation of both rotavirus and calicivirus within the Maternity Hospital. It is possible that the interruption of rotavirus transmission within this Institution could be due to changes in the management of the mother-child unit promoted by WHO and UNICEF, and introduced at the Maternity Hospital since 1995.

Evaluation of serum antibody responses against the rotavirus nonstructural protein NSP4 in children after rhesus rotavirus tetravalent vaccination or natural infection.

The immune response elicited by the rotavirus nonstructural protein NSP4 and its potential role in protection against rotavirus disease are not well understood. We investigated the serological response to NSP4 and its correlation with disease protection in sera from 110 children suffering acute diarrhea, associated or not with rotavirus, and from 26 children who were recipients of the rhesus rotavirus tetravalent (RRV-TV) vaccine. We used, as antigens in an enzyme-linked immunosorbent assay (ELISA), affinity-purified recombinant NSP4 (residues 85 to 175) from strains SA11, Wa, and RRV (genotypes A, B, and C, respectively) fused to glutathione S-transferase. Seroconversion to NSP4 was observed in 54% (42/78) of the children who suffered from natural rotavirus infection and in 8% (2/26) of the RRV-TV vaccine recipients. Our findings indicate that NSP4 evokes significantly (P < 0.05) higher seroconversion rates after natural infection than after RRV-TV vaccination. The serum antibody levels to NSP4 were modest (titers of < or = 200) in most of the infected and vaccinated children. A heterotypic NSP4 response was detected in 48% of the naturally rotavirus-infected children with a detectable response to NSP4. Following natural infection or RRV-TV vaccination, NSP4 was significantly less immunogenic than the VP6 protein when these responses were independently measured by ELISA. A significant (P < 0.05) proportion of children who did not develop diarrhea associated with rotavirus had antibodies to NSP4 in acute-phase serum, suggesting that serum antibodies against NSP4 might correlate with protection from rotavirus diarrhea. In addition, previous exposures to rotavirus did not affect the NSP4 seroconversion rate.