Humoral Immunity across the SARS-CoV-2 Spike after Sputnik V (Gam-COVID-Vac) Vaccination.

SARS-CoV-2 vaccines have contributed to attenuating the burden of the COVID-19 pandemic by promoting the development of effective immune responses, thus reducing the spread and severity of the pandemic. A clinical trial with the Sputnik-V vaccine was conducted in Venezuela from December 2020 to July 2021. The aim of this study was to explore the antibody reactivity of vaccinated individuals towards different regions of the spike protein (S). Neutralizing antibody (NAb) activity was assessed using a commercial surrogate assay, detecting NAbs against the receptor-binding domain (RBD), and a plaque reduction neutralization test. NAb levels were correlated with the reactivity of the antibodies to the spike regions over time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the virus during the clinical trial in the serological response. A high serological reactivity was observed to S and specifically to S1 and the RBD. S2, although recognized with lower intensity by vaccinated individuals, was the subunit exhibiting the highest cross-reactivity in prepandemic sera. This study is in agreement with the high efficacy reported for the Sputnik V vaccine and shows that this vaccine is able to induce an immunity lasting for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed us to identify the relevance of epitopes outside the RBD that are able to induce NAbs. This research may contribute to the understanding of vaccine immunity against SARS-CoV-2, which could contribute to the design of future vaccine strategies.

Neutralization of Different Variants of SARS-CoV-2 by a F(ab')2 Preparation from Sera of Horses Immunized with the Viral Receptor Binding Domain.

The Receptor Binding Domain (RBD) of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, is the functional region of the viral Spike protein (S), which is involved in cell attachment to target cells. The virus has accumulated progressively mutations in its genome, particularly in the RBD region, many of them associated with immune evasion of the host neutralizing antibodies. Some of the viral lineages derived from this evolution have been classified as Variant of Interest (VOI) or Concern (VOC). The neutralizing capacity of a F(ab')2 preparation from sera of horses immunized with viral RBD was evaluated by lytic plaque reduction assay against different SARS-CoV-2 variants. A F(ab')2 preparation of a hyperimmune serum after nine immunizations with RBD exhibited a high titer of neutralizing antibodies against the ancestral-like strain (1/18,528). A reduction in the titer of the F(ab')2 preparation was observed against the different variants tested compared to the neutralizing activity against the ancestral-like strain. The highest reduction in the neutralization titer was observed for the Omicron VOC (4.7-fold), followed by the Mu VOI (2.6), Delta VOC (1.8-fold), and Gamma VOC (1.5). Even if a progressive reduction in the neutralizing antibodies titer against the different variants evaluated was observed, the serum still exhibited a neutralizing titer against the Mu VOI and the Omicron VOC (1/7113 and 1/3918, respectively), the evaluated strains most resistant to neutralization. Therefore, the preparation retained neutralizing activity against all the strains tested.

Hepatitis B eradication: vaccine as a key player.

OBJECTIVE: Despite the availability of a highly effective and safe vaccine against hepatitis B virus (HBV) infection for 40 years, still almost 300 million persons are estimated to be chronically infected by this virus worldwide. The World Health Organization (WHO) has proposed a plan for hepatitis elimination by 2030. However, several factors, such as the reduction and limitation in vaccination campaigns or vaccine hesitancy (VH) in some regions of the World, might have played a role in limiting the worldwide coverage of hepatitis B prophylaxis. This review aims to describe which factors, such as VH, may be hampering the WHO 2030 goal for hepatitis B eradication. METHODS: The review describes the development and characteristics of the HBV vaccine, from the first plasma-derived to the recombinant one. Eventual limitations in its effectiveness and particularly VH were reviewed. RESULTS: The apparent pitfalls of the HBV vaccine, such as long-term effectiveness, vaccine-escape mutants, and adverse effects, were proven not to be a concern for this vaccine. However, VH persists and was even intensified by the COVID-19 pandemic. CONCLUSIONS: Many barriers still exist, such as vaccine availability, lack of awareness of the benefits of HBV vaccination, and VH. HBV VH seems to be eventually overcome in many settings with active education campaigns and information, stressing the importance of developing these strategies to achieve the 2030 goal of the WHO.

Antiviral activity of flavonoids present in aerial parts of Marcetia taxifolia against Hepatitis B virus, Poliovirus, and Herpes Simplex Virus in vitro.

Marcetia taxifolia is a neotropical plant present in South America and it has been evaluated in several biological models due to the presence of active metabolites. Nevertheless, there is a limited quantity of studies related to the antiviral activity of the compounds present in this genus. In our work, the antiviral effect of the compounds isolated from the aerial parts of Marcetia taxifolia was evaluated against Hepatitis B virus (HBV), Herpes Simplex Virus type 1 (HSV-1), and Poliovirus type 1 (PV-1). The cytopathic effect and viral quantification by qPCR were determined as indicative of antiviral activity. Our data show that myricetin rhamnoside (MyrG), myricetin-3-α-O-ramnosil (1→6)-α-galactoside (MyrGG), 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxyflavone (PMF), 5-hydroxy-3,6,7,3',4'pentamethoxyflavone (PMF-OH) had antiviral activity without cytotoxic effects. The methoxyflavones PMF and PMF-OH were the most active compounds, showing an antiviral effect against all the evaluated viruses. Computational studies showed that these compounds could interact with the Reverse Transcriptase. Altogether, these results suggest that the flavonoids (related to myricetin and methoxyflavones) are the main antiviral compounds present in the aerial parts of Marcetia taxifolia. Furthermore, our results showed that the methoxyflavones have a broad antiviral activity, which represents an opportunity to evaluate these flavonoids as lead molecules to develop new antiviral compounds.

Performance of molecular methods for identification of unusual subtypes of hepatitis C virus genotype 2

Introduction: Hepatitis C virus (HCV) displays high genetic variability, with seven genotypes and numerous subtypes. The determination of the viral type has been essential for the selection and timing of antiviral treatment. In Venezuela, HCV genotype 2 is relatively diverse, being particularly prevalent subtype 2j. Objective: To evaluate the performance of methodologies for genotyping HCV, particularly for identification of subtype 2j. Materials and methods: HCV genotype and subtype were determined by reverse hybridization technique (LiPA) and sequencing of the HCV 5'UTR and NS5B regions. Results: A total of 65 samples from HCV-infected patients were analyzed. PCR amplifications of the 5'UTR region exhibited the highest sensitivity (100% vs 91% for LiPA and 77% for NS5B). Genotype determination, taking as reference test NS5B, showed 100% concordance with the other methods, and 67% and 59% for subtypes with 5´NC and LiPA, respectively. NS5B sequencing allowed the identification of subtypes 2j and 2s, which were not detected by the other methods. A specific LiPA pattern was not observed for HCV subtype 2j. Conclusion: Although being the methodology with lowest sensitivity for amplification of HCV RNA, sequencing NS5B region remains a powerful tool for correct discrimination of the different HCV subtypes, which is of epidemiological relevance.

Desempeño de métodos moleculares para la identificación de subtipos poco comunes del virus de la hepatitis C, genotipo 2 / Performance of molecular methods for identification of unusual subtypes of hepatitis C virus genotype 2

Resumen Introducción. El virus de la hepatitis C (HCV) presenta una gran variabilidad genética, con siete genotipos y numerosos subtipos. La determinación del tipo viral ha sido fundamental para la escogencia y la duración del tratamiento antiviral adecuado. En Venezuela, el genotipo 2 del HCV es relativamente diverso, siendo particularmente prevalente el subtipo 2j. Objetivo. Evaluar el desempeño de las metodologías para la determinación del genotipo del HCV, particularmente para la identificación del subtipo 2j. Materiales y métodos. Se determinaron el genotipo y el subtipo del HCV mediante la técnica de hibridación inversa LiPA (Line Probe Assay) y secuenciación de las regiones genómicas 5'NC y NS5B del virus. Resultados. En 65 muestras analizadas, la metodología basada en la amplificación de la región 5'NC mostró mayor sensibilidad (100 %), en comparación con la técnica LiPA (91 %) y la secuenciación de la región NS5B (77 %). La determinación de genotipo, tomando como método de referencia la secuenciación de NS5B, mostró un alto grado de concordancia para la secuenciación de la región 5´NC y la hibridación inversa LiPA, con 100 % en la asignación de genotipos, comparado con 70 % y 66 % para los subtipos, respectivamente. La secuenciación de la región NS5B permitió identificar los subtipos 2j y 2s, los cuales no fueron detectados por las otras metodologías. No se observó un patrón característico para las muestras subtipo 2j en la hibridación inversa LiPA. Conclusión. Aunque es la metodología con menor sensibilidad, la secuenciación de la región NS5B es una herramienta poderosa para la correcta discriminación de los distintos subtipos circulantes del HCV, lo cual reviste importancia epidemiológica.

Abstract Introduction: Hepatitis C virus (HCV) displays high genetic variability, with seven genotypes and numerous subtypes. The determination of the viral type has been essential for the selection and timing of antiviral treatment. In Venezuela, HCV genotype 2 is relatively diverse, being particularly prevalent subtype 2j. Objective: To evaluate the performance of methodologies for genotyping HCV, particularly for identification of subtype 2j. Materials and methods: HCV genotype and subtype were determined by reverse hybridization technique (LiPA) and sequencing of the HCV 5'UTR and NS5B regions. Results: A total of 65 samples from HCV-infected patients were analyzed. PCR amplifications of the 5'UTR region exhibited the highest sensitivity (100% vs 91% for LiPA and 77% for NS5B). Genotype determination, taking as reference test NS5B, showed 100% concordance with the other methods, and 67% and 59% for subtypes with 5´NC and LiPA, respectively. NS5B sequencing allowed the identification of subtypes 2j and 2s, which were not detected by the other methods. A specific LiPA pattern was not observed for HCV subtype 2j. Conclusion: Although being the methodology with lowest sensitivity for amplification of HCV RNA, sequencing NS5B region remains a powerful tool for correct discrimination of the different HCV subtypes, which is of epidemiological relevance.

Mutaciones K130M y V131I del gen X en aislados venezolanos del virus de hepatitis B / K130M and V131L Mutations on the X gene of hepatitis B virus in Venezuelan isolates

Aproximadamente el 50% de los carcinomas hepatocelulares (CHC) en el mundo están etiológicamente asociados con la infección por el virus de hepatitis B (VHB). Se han descrito 10 genotipos del VHB (A-J). En Venezuela y en varios países latinoamericanos predomina el genotipo F. Las mutaciones K130M y V131I presentes en la proteína HBx del VHB han sido asociadas al desarrollo del CHC. El objetivo de este trabajo fue estudiar la variabilidad genética de la proteína HBx del VHB circulante en pacientes venezolanos, con el fin de correlacionar estas mutaciones con los parámetros clínicos y virológicos de la enfermedad. Se analizó la secuencia del gen X del VHB, mediante amplificación por PCR de un fragmento de ese gen, en 45 pacientes infectados (35 crónicos y 10 agudos). Se observó una mayor frecuencia de las mutaciones K130M y V131I en pacientes de 25 o más años y con infección crónica. La presencia de estas mutaciones fue significativamente menor en el subgenotipo F3, comparado con el genotipo C. Estos resultados refuerzan la hipótesis de que el subgenotipo F3, predominante en Venezuela, podría estar asociado a una progresión menos severa de la enfermedad que la descrita para otros subgenotipos americanos, como F1b o F2.

Approximately 50% of the hepatocellular carcinomas (HCC) in the world are etiologically associated to hepatitis B virus (HBV) infection. Ten HBV genotypes (A-J) have been described in Venezuela and in other Latin American countries where the F genotype predominates. The K130M and V131I mutations present in the HBx protein of HBV have been associated with the development of HCC. The aim of this work was to study the genetic variability of HBx protein from HBV circulating in Venezuelan patients, in order to correlate these mutations with clinical and virus factors involved in the disease. The X HBV gene sequence was analyzed by PCR amplification of that gene in 45 infected patients (35 with chronic and 10 with acute stages of hepatitis). A higher frequency K130M and V131I mutations was observed in subjects 25 years of age and older with chronic infection. The presence of these mutations was significantly lower in the F3 subgenotype compared with genotype C. These results support the hypothesis that the F3 subgenotype, predominant in Venezuela, could be associated with a less severe progression of the disease than that described for other American subgenotypes, such as F1b or F2.

Infección por el virus de la hepatitis C (HCV) en pacientes hemodializados seronegativos para anticuerpos anti-HCV / Hepatitis C virus (HCV) infection in hemodialysed patients seronegative for anti-HCV antibodies

La infección por el virus de la hepatitis C (HCV) es común en pacientes hemodializados. Se evaluaron 43 sueros de pacientes de la Unidad de Diálisis “Dr. José Maza Carvajal” del Servicio Autónomo del Hospital Universitario “Antonio Patricio de Alcalᔠ(SAHUAPA), en Cumaná, estado Sucre. Se determinaron anticuerpos IgG séricos contra el HCV (anti-HCV) utilizando tres técnicas inmunoenzimáticas. Para amplificar la región 5’ no codificante (5’NC) se usó la técnica de transcripción reversa de la reacción en cadena de la polimerasa (RT-PCR), en muestras positivas y negativas para anti-HCV. La presencia de anticuerpos y RNA del HCV fue de 9,3% y la presencia de RNA del HCV en pacientes con anti-HCV negativos fue de 42%, lo cual representó una frecuencia de infección activa de 51%. Análisis filogenéticos de la región 5’NC evidenciaron que el genotipo 2 fue el más prevalente, en particular el subtipo 2b, seguido por el genotipo 1, mientras que en siete muestras no se logró identificar el subtipo. La presencia de un alto número de pacientes seronegativos e infectados con el HCV puede deberse al estado de inmunocompromiso de estos pacientes; de allí la importancia de la determinación de la viremia.

Hepatitis C virus infection is common in hemodialysed patients. Sera from 43 patients from the Dialysis Unit “Dr. José Maza Carvajal” of the University Hospital “Antonio Patricio de Alcalá”, in Cumana, Sucre state, were evaluated. Antibodies against HCV (anti-HCV) were determined using three immunosorbent assays. The 5 ‘non-coding (5’NC) HCV region was amplified by RT-PCR in all samples. The presence of antibodies and HCV RNA was 9.3% and of HCV RNA in seronegative sera 42%, which represents a frequency of infection of 51%. Phylogenetic analysis of the 5’NC region showed that genotype 2 was the most frequently found, particularly due to subtype 2b, followed by genotype 1, while seven subtypes could not be determined. The presence of a high number of seronegative HCV-infected hemodialysed patients might be due to the immunocompromised condition of these patients; hence the importance of determining the viremia.

HIV-1 and GBV-C co-infection in Venezuela.

INTRODUCTION: Co-infection with GB virus C (GBV-C) in patients infected with human immunodeficiency virus 1 (HIV-1) has been associated with prolonged survival. The aim of this study was to evaluate the prevalence of GBV-C infection among HIV-1-infected patients in Venezuela, and to determine the effects of the co-infection on the levels of relevant cytokines. METHODOLOGY: Plasma samples were collected from 270 HIV-1-seronegative and 255 HIV-1-seropositive individuals. GBV-C infection was determined by RT-PCR of the NS5 region and genotyped by sequence analysis of the 5´UTR region. HIV-1 strains were characterized by sequence analysis of pol, vif, env, and nef genes. Selected cytokines were evaluated by ELISA. RESULTS: Ninety-seven of 525 (18.5%) plasma samples tested positive for GBV-C RNA. A significantly higher prevalence of GBV-C was found among HIV-1 patients compared to HIV-1-seronegative individuals (67/255, 26% versus 30/270, 11%; p < 0.001). Statistical difference was observed in the viral load between HIV-1+GBV-C+ and HIV-1+GBV-C- (p = 0.014), although no differences in CD4+ cell counts were found between both groups. TNFα concentration was higher in HIV-1+GBV-C- than in HIV-1+GBV-C+ patients (25.9 pg/mL versus 17.3 pg/mL; p = 0.02); RANTES expression levels were more variable in GBV-C co-infected patients and more frequently elevated in HIV-1 mono-infected patients compared to patients co-infected with GBV-C. CONCLUSIONS: The previously observed beneficial effect of co-infection with HIV-1 and GBV-C on disease progression is complex and might be due in part to a change in the cytokine environment. More studies are required to understand the interaction between both viruses.

Molecular characterization of hepatitis E virus in patients with acute hepatitis in Venezuela.

Hepatitis E virus (HEV) causes a common infection in developing countries. HEV infection occurs as outbreaks, as sporadic clinical cases and as large epidemics in endemic areas. The objective of this study was to determine the presence of HEV infection in patients with clinical suspicion of hepatitis A virus (HAV) infection, referred to the Instituto Nacional de Higiene "Rafael Rangel" in Venezuela. Seventy-four sera were tested for anti-HAV and anti-HEV IgM antibodies. HEV-RNA was amplified from anti-HEV IgM positive sera using nested reverse transcription polymerase chain reaction for ORF1 (RNA dependent RNA polymerase region) and the amplicons sequenced for phylogenetic analysis. The frequency of anti-HEV IgM was 22/74 (30%) in the samples tested. Dual infection with HAV and HEV was found in 31% (12/39) of anti-HAV IgM positive patients. Viremia was detected in 3/22 (14%) of sera positive for anti-HEV IgM. Two HEV strains were classified as genotype 1 and one as genotype 3, which were closely related to Yam 67 (north of India) and US1 isolates from the USA, respectively. These findings suggest that HEV is an important cause of acute viral hepatitis in Venezuela as a single infection or co-infection with HAV, with high morbidity in children and young adults suggesting that this infection is endemic in Venezuela.

Human papillomavirus in invasive cervical cancer and cervical intraepithelial neoplasia 2 and 3 in Venezuela: a cross-sectional study.

BACKGROUND: This study investigated the distribution of human papillomavirus (HPV) types in invasive cervical cancer (ICC), cervical intraepithelial neoplasia 2 (CIN2) and cervical intraepithelial neoplasia 3 (CIN3) in Venezuela. METHODS: Paraffin-embedded samples from 329 women from 29 medical centers of the 24 states of Venezuela were analyzed to determine the distribution of HPV types for ICC, CIN2, and CIN3, the prevalence of single and multiple infection, and the association of HPV types with severity of lesion, comparing CIN2 versus CIN3+ (CIN3 and ICC). The samples were analyzed with the polymerase chain reaction (PCR) followed by reverse hybridization for the identification of HPV types. RESULTS: HPV was identified in 95/96 ICC specimens (98.9%), in 142/149 CIN3 (95.3%) and in 78/84 CIN2 samples (92.8%). The most common types for ICC and CIN3 were: HPV16, 18, 31, and 33, and for CIN2 were HPV16, 31, 51, 52, and 18. HPV single infection was found in 82.1% of ICC cases, in 79.4% of CIN2 cases, and in 77.4% of CIN3 cases. HPV16 was identified as a single infection more frequently in women with CIN3+ than in those with CIN2 (68.6% versus 46.7%, P=0.002), and HPV16 or HPV18 types were more prevalent in CIN3+ than in CIN2 (73.4% versus 50%, P=0.0006). CONCLUSION: this is the first study of the distribution of HPV types in ICC, CIN2, and CIN3 conducted throughout the territory of Venezuela. HPV16 and HPV18 were the most frequent HPV types identified in single and multiple infections in both ICC and CIN3 groups, and are associated with severity of lesion. The knowledge of the distribution of HPV types would allow organization of an HPV-DNA-based screening test, and consideration of the implementation of prophylactic vaccination in Venezuela.

Etiology and Viral Genotype in Patients with End-Stage Liver Diseases admitted to a Hepatology Unit in Colombia.

Hepatitis B virus (HBV) and hepatitis C virus (HCV) infections are the principal risk factor associated to end-stage liver diseases in the world. A study was carried out on end-stage liver disease cases admitted to an important hepatology unit in Medellin, the second largest city in Colombia. From 131 patients recruited in this prospective study, 71% of cases were diagnosed as cirrhosis, 12.2% as HCC, and 16.8% as cirrhosis and HCC. Regarding the risk factors of these patients, alcohol consumption was the most frequent (37.4%), followed by viral etiology (17.6%). Blood and/or hepatic tissue samples from patients with serological markers for HCV or HBV infection were characterized; on the basis of the phylogenetic analysis of HCV 5' UTR and HBV S gene, isolates belonged to HCV/1 and HBV/F3, respectively. These results confirm the presence of strains associated with poor clinical outcome, in patients with liver disease in Colombia; additionally, HBV basal core promoter double mutant was identified in HCC cases. Here we show the first study of cirrhosis and/or HCC in Colombian and HBV and HCV molecular characterization of these patients. Viral aetiology was not the main risk factor in this cohort but alcohol consumption.

Desempeño de estuches comerciales para el diagnóstico serológico de las hepatitis virales B y C en poblaciones yanomami y piaroa del estado Amazonas / Performance of commercial kits for serologic diagnosis of viral hepatitis B and C in yanomami and piaroa populations from Amazonas State

Los virus de hepatitis son una causa importante de morbilidad y mortalidad en la cuenca amazónica. El objetivo de este estudio fue evaluar el desempeño de estuches serológicos para la determinación de marcadores de VHB y VHC en población indígena. Se determinó la presencia de anti-HBc, agsHB, anti-VHC y de genomas virales en sueros de individuos piaroa y yanomami. Más de 50% de las muestras reactivas por un inmunoensayo comercial no resultaron positivas al usar otros estuches. El marcador serológico para el cual se observó una mayor concordancia entre los estuches comerciales fue el anti-HBc, posiblemente porque se trata de un ensayo de inhibición. La concordancia entre los ensayos para agsHB con la positividad de la PCR fue de pobre a moderada, coincidiendo sólo dos de los ensayos con los resultados de la PCR. No existió concordancia entre los distintos ensayos inmunoezimáticos, ni con la presencia del ARN viral para VHC. Las discrepancias inesperadas entre distintos estuches comerciales pudieran deberse a características inherentes a estas poblaciones, tales como múltiples coinfecciones, en especial parasitarias. Estos factores pudiesen estar afectando la especificidad de los estuches diagnósticos, situación observada con menor frecuencia en otras poblaciones venezolanas. Estos estudios refuerzan la importancia de la validación de pruebas serológicas en estas poblaciones, con ensayos confirmatorios y moleculares.

Hepatitis viruses are an important morbility and mortality cause in the Amazon basin. The goal of this study was to evaluate the performance of commercial serologic kits for determination of HVB and HVC markers in indigenous populations. Presence of anti-HBc, HBags, anti-HVC and viral genomes was determined in sera from piaroa and yanomami individuals. Over 50% of the samples reactive with one of the commercial kits were not positive when using other kits. The serologic marker which showed the highest concordance among the commercial kits was anti-HBc, possibly because it is an inhibition assay. Concordance among assays for HBags and PCR positivity varied between poor and moderate; only two of the tests coincided with the PCR results. There was no concordance among the various immunoenzymatic assays, nor in viral RNA presence for HVC. The unexpected discrepancies among the various commercial kits could be due to inherent characteristics of these populations such as multiple co-infections, especially parasitic. These factors could be affecting the specificity of the diagnostic kits, situation less frequently observed in other Venezuelan populations. This study emphasizes the importance of validating serologic tests in these populations, through confirmation and molecular assays.

Deletion, insertion and stop codon mutations in vif genes of HIV-1 infecting slow progressor patients.

BACKGROUND: Variable progression towards AIDS has been described and has been related to viral and host factors. Around 10% of the HIV-1 infected patients are slow progressors (SP), not presenting with AIDS disease signs even after more than 10 years of infection. Viral gene defects have been associated with the disease progression but more studies are still needed. METHODOLOGY: The sequence of vif and nef were analyzed for HIV-1 infecting 14 SP and 46 normal progressors (NP) patients. RESULTS: Co-circulation of a strain carrying vif deleted gene with the wild type strain was detected in an SP patient with more than 10 years of infection. Other mutations (insertion in aa 63 in one strain, two premature stop codons in another one) were found in viruses infecting two other patients. Except for the SP8 strain, which exhibited a premature stop codon in nef, no gross deletions or insertions were observed in nef genes of both NPs and SPs strains analyzed. CONCLUSIONS: Different kind of mutation: deletion, insertion and stop codon, were detected in 3/14 samples from SP, with co-circulation of a 195 bp vif deletion virus with a wild type in one of these patients. Although vif defects do not seem to be a frequent feature in SPs, this study illustrates the importance of analysing this gene, in addition to the multiple factors associated with the long-term non progression to AIDS.

Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma.

Hepatitis B viruses (HBV) are responsible for over 50% of the worldwide attributable risk of hepatocellular carcinoma (HCC) and this figure increases even further in regions of high endemicity. Systematic sequencing of HBV genomes has identified that this common virus existed as eight distinct genotypes (denoted A-H), each regrouping variants with less than 8% divergence in their DNA sequence. These genotypes differ by their geographic distribution in populations around the globe. There is evidence that HBV genotypes also differ by their pathogenic properties, including their risk of persistence as chronic infection and their capacity to induce precursor disease or cancer. On the other hand, HBV genes may undergo mutations that become selected during the course of chronic infection and progressive liver disease. The most significant of these mutations in the context of HCC are those occurring in the pre-core (Pre-C) and basal core promoter (BCP) regions. These mutations may upregulate HBV expression and increase its virulence. These mutations may occur in all HBV genotypes but are more common in genotypes associated with more severe disease and cancer, in particular genotype C. Understanding the molecular basis of pathological variations between HBV variants is critical for prediction of disease severity. It will also be important to determine whether differences among genotypes may have an impact on the long-term protective efficacy of universal HBV vaccination.

Prevalence of antiretroviral drug resistance among treatment-naive and treated HIV-infected patients in Venezuela.

An in-house, low-cost method was developed to determine the genotypic resistance of immunodeficiency virus type 1 (HIV-1) isolates. All 179 Venezuelan isolates analysed belonged to subtype B. Primary drug resistance mutations were found in 11% of 63 treatment-naïve patients. The prevalence of resistance in isolates from 116 HIV-positive patients under antiretroviral treatment was 47% to protease inhibitors, 65% to nucleoside inhibitors and 38% to non-nucleoside inhibitors, respectively. Around 50% of patients in the study harboured viruses with highly reduced susceptibility to the three classical types of drugs after only five years from their initial diagnoses.

Prevalence of antiretroviral drug resistance among treatment-naive and treated HIV-infected patients in Venezuela

An in-house, low-cost method was developed to determine the genotypic resistance of immunodeficiency virus type 1 (HIV-1) isolates. All 179 Venezuelan isolates analysed belonged to subtype B. Primary drug resistance mutations were found in 11 percent of 63 treatment-naïve patients. The prevalence of resistance in isolates from 116 HIV-positive patients under antiretroviral treatment was 47 percent to protease inhibitors, 65 percent to nucleoside inhibitors and 38 percent to non-nucleoside inhibitors, respectively. Around 50 percent of patients in the study harboured viruses with highly reduced susceptibility to the three classical types of drugs after only five years from their initial diagnoses.

High prevalence of secondary resistance mutations in Venezuelan HIV-1 isolates.

The genetic variability was studied in HIV-1 from Venezuelan patients with and without treatment, in order to evaluate the presence of polymorphisms and drug resistance mutations. Proviral DNA from peripheral blood mononuclear cells or viral RNA from plasma was extracted from the blood of 30 patients. Two regions from the polymerase gene, protease (Pr) and reverse transcriptase (RT) and one genomic fragment from the envelope (Env) gene were amplified and sequenced. All HIV-1 samples analyzed were classified as subtype B, without evidence of recombination. Although no primary protease mutations were detected, a high frequency of secondary mutations (86%, 19/22), associated to restoration of viral replicative fitness, was observed in strains circulating both in treated and non-treated patients. Resistance mutations to nucleoside RT inhibitors (NRTI) and non-nucleoside RT inhibitors (NNRTI) were detected in 35% (6/17) and 12% (2/17) of the viruses circulating in treated patients, respectively. Resistance mutations were also present in the virus infecting one antiretroviral naive individual (7.7%), suggesting that local screening for resistant mutation in naive patient might be important to minimize therapy failure. Future studies are warranted to assess the role of secondary mutation in the success of viral infection.

High prevalence of secondary resistance mutations in Venezuelan HIV-1 isolates

Se estudiaron pacientes seropositivos para el virus de inmunodeficiencia humana tipo 1 (VIH-1) con y sin tratamiento, con el fin de determinar el polimorfismo y la prevalencia de mutaciones de resistencia a la terapia antirretroviral. El material genético viral fue extraído a partir de células mononucleares de sangre periféricas (ADN) y del plasma (ARN) de 30 pacientes. Se amplificaron 2 regiones del gen Pol, Transcriptasa Reversa (TR) y Proteasa (Pr) y el gen de envoltura (Env) por medio de la técnica de PCR y se obtuvo la secuencia genómica de los productos. Todos los aislados analizados pertenecieron al subtipo B. No se observaron mutaciones primarias asociadas a resistencia a inhibidores de Pr pero sí un alto porcentaje (86 por ciento, 19/22) de mutaciones no asociadas con resistencia sino a restitución de la capacidad replicativa de cepas mutantes (mutaciones secundarias). Se observó la presencia de mutaciones asociadas a resistencia a inhibidores nucleósidos de la TR (INTR) en 35 por ciento (6/17) de los pacientes sometidos a tratamiento, mientras que 12 por ciento (2/17) de ellos presentaron mutaciones de resistencia a inhibidores no nucleósidos de la TR (INNTR). Interesantemente, un paciente no tratado estaba infectado con una cepa que presentaba mutaciones primarias (7,7 por ciento); este resultado sugiere que podría ser importante plantearse el estudio local de determinación de resistencia genotípica en pacientes antes del tratamiento, con miras a minimizar fallas terapéuticas. Se requieren estudios adicionales para evaluar el rol de las mutaciones secundarias en el éxito de la infección viral