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.

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.

[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.

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.

[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.

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.

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.

HUMAN COSAVIRUS INFECTION IN HIV SUBJECTS WITH DIARRHOEA: PERSISTENT DETECTION ASSOCIATED WITH FATAL OUTCOME.

BACKGROUND: Human cosavirus (HCoSV) is a new member of the Picornaviridae family, geographically widespread among humans. It has been suggested as a causative agent of acute gastroenteritis, but its pathogenicity is not currently certain. In HIV-infected subjects, diarrhoea is one of the most frequent gastrointestinal manifestations, whose aetiology remains often unexplained. OBJECTIVES: To identify the cause of viral diarrhoea among HIV infected patients by molecular assays. STUDY DESIGN: A total of 143 stool samples from HIV subjects with and without diarrhoea, were screened for conventional enteric viruses (rotavirus, adenovirus, norovirus and astrovirus) by molecular assays. The presence of HCoSV genome was investigated by nested RT-PCR for the 5'UTR region. Positive samples were further characterized by sequencing and phylogenetic analysis. RESULTS: Enteric viruses were more frequently found in diarrhoea cases (9/82) than controls (0/61) (p=0.007). HCoSV was detected in five (3.5%) of the subjects affected by diarrhoea. Phylogenetic analysis revealed the predominance of the HCoSV species D. One patient suffered a persistent cosavirus infection with a same strain and after eight months he had a fatal outcome. No other pathogens could be detected. CONCLUSIONS: The results suggest a role of non-conventional enteric viruses, as HCoSV, as a potential opportunistic agent causing persistent infection and deterioration of the clinical conditions in HIV-infected patients. Screening procedures and monitoring including such viruses would be helpful in the clinical management of such patients.

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 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.