Salvador Fonseca-Coronado

Specific Detection of Naturally Occurring Hepatitis C Virus Mutants with Resistance to Telaprevir and Boceprevir (Protease Inhibitors) among Treatment-Naïve Infected Individuals

ABSTRACT The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of selective pressure, drug-resistant hepatitis C virus (HCV) mutants are generally present at low frequency, making mutation detection challenging. Here, we describe a mismatch amplification mutation assay (MAMA) PCR method for the specific detection of naturally occurring drug-resistant HCV mutants. MAMA PCR successfully identified the corresponding HCV variants, while conventional methods such as direct sequencing, endpoint limiting dilution (EPLD), and bacterial cloning were not sensitive enough to detect circulating drug-resistant mutants in clinical specimens. Ultradeep pyrosequencing was used to confirm the presence of the corresponding HCV mutants. In treatment-naïve patients, the frequency of all resistant variants was below 1%. Deep amplicon sequencing allowed a detailed analysis of the structure of the viral population among these patients, showing that the evolution of the NS3 is limited to a rather small sequence space. Monitoring of HCV drug resistance before and during treatment is likely to provide important information for management of patients undergoing anti-HCV therapy.

Interleukin-28B Genotyping by Melt-Mismatch Amplification Mutation Assay PCR Analysis Using Single Nucleotide Polymorphisms rs12979860 and rs8099917, a Useful Tool for Prediction of Therapy Response in Hepatitis C Patients

ABSTRACT Several studies have identified associations between single nucleotide polymorphisms (SNPs) occurring near the interleukin-28B (IL-28B) gene and response to antiviral treatment among hepatitis C virus (HCV) patients. Here, we describe a reliable melt-mismatch amplification mutation assay (melt-MAMA) PCR-based genotyping method for IL-28B which can be used in the management of HCV patients, helping to better define the course of therapy.

Hepatitis C virus molecular evolution: transmission, disease progression and antiviral therapy.

Hepatitis C virus (HCV) infection represents an important public health problem worldwide. Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors. Virus molecular evolution plays an important role in HCV transmission, disease progression and therapy outcome. The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures (e.g., host immune responses and antiviral therapy). HCV molecular evolution is shaped by different mechanisms including a high mutation rate, genetic bottlenecks, genetic drift, recombination, temporal variations and compartmentalization. These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner. Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV. As a result, superior sustained viral responses have been attained. The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer, more potent antivirals, bringing us one step closer to the interferon-free era.

Rapid Hepatitis C Virus Divergence among Chronically Infected Individuals

ABSTRACT Here, we analyze the viral divergence among hepatitis C virus (HCV) chronic cases infected with genotype 1. The intrahost viral evolution was assessed by deep sequencing using the 454 Genome Sequencer platform. The results showed a rapid nucleotide sequence divergence. This notorious short-term viral evolution is of the utmost importance for the study of HCV transmission, because direct links between related samples were virtually lost. Thus, rapid divergence of HCV significantly affects genetic relatedness studies and outbreak investigations.

Taenia solium: Immune response against oral or systemic immunization with purified recombinant calreticulin in mice

Recombinant functional Taenia solium calreticulin (rTsCRT) confers different degrees of protection in the experimental model of intestinal taeniosis in hamsters. The aim of this study was to evaluate the immune response induced after oral or systemic immunization with an electroeluted rTsCRT in BALB/c mice. Oral immunization elicited high fecal IgA and the production of IL-4 and IL-5 by mesenteric lymph node cells after in vitro stimulation with rTSCRT, indicating a Th2 response. Mice subcutaneously immunized produced high amounts of serum IgG, being IgG1 (Th2-related) the predominant isotype, while in vitro stimulated spleen cells synthesized IL-4, IL-5 and also IFN-γ, indicating a mixed Th1/Th2 cellular response after systemic immunization. Our data show that purified rTsCRT induces polarized Th2 responses after oral immunization of mice, a common characteristic of protective immunity against helminths and, consequently, a desirable hallmark in the search for a vaccine.

Molecular epidemiology of viral diseases in the era of next generation sequencing.

In virology, molecular epidemiology is defined as the applica-tion of molecular methods to study the causation and dynamicsof viral diseases in a population. In the study of viral diseases,this is determined by the transmission of the corresponding eti-ological agent on its host, and how this phenomenon affects thespread of the disease within the population. Molecular epidemi-ology uses information about genetic variation to investigate theevolution, transmissionandpathogenesisofviralagents.Molecularepidemiological toolshaveprovidedimportantinsightsinthestudyof viral diseases, improving significantly our understanding aboutthe history of infection, virus evolution, spread and disease trans-mission that have helped to implement public health policies.

Epidemiology of varicella in Mexico

BACKGROUND The epidemiological patterns of varicella-zoster virus (VZV) infection, which are strongly associated with climate, are characterized by more frequent infections occurring among children in temperate regions than in the tropics. In temperate regions, varicella exhibits a seasonal cyclic behavior in which the number of cases increases significantly during the winter and spring seasons, further supporting the role of environmental factors in disease transmission. However, the underlying mechanisms responsible for this distinctive behavior are not fully understood. In Mexico, information regarding the epidemiology of varicella is scarce, and the distribution of VZV infection has not been analyzed. OBJECTIVES In this article we investigate the epidemiological patterns of varicella in Mexico and their relationship with different environmental and demographic factors. STUDY DESIGN A retrospective study was conducted using the data reported by the National Center of Epidemiological Surveillance and Disease Control. The overall varicella incidence was calculated and associated with temperature, overcrowding, age, gender and population density. RESULTS The epidemiology of varicella showed an intriguing pattern, in which warmer regions were characterized by higher incidences than in temperate regions. Young children were the most affected age group. There was no correlation between varicella incidence and overcrowding or population density. CONCLUSIONS The epidemiology of varicella in Mexico significantly departs from the characteristic patterns observed in other tropical latitudes, with some features resembling those commonly associated with temperate regions.

Vertical transmission of hepatitis C virus: a tale of multiple outcomes.

Globally, hepatitis C virus (HCV) infection affects approximately 130 million people and 3 million new infections occur annually. HCV is also recognized as an important cause of chronic liver disease in children. The absence of proofreading properties of the HCV RNA polymerase leads to a highly error prone replication process, allowing HCV to escape host immune response. The adaptive nature of HCV evolution dictates the outcome of the disease in many ways. Here, we investigated the molecular evolution of HCV in three unrelated children who acquired chronic HCV infection as a result of mother-to-child transmission, two of whom were also coinfected with HIV-1. The persistence of discrete HCV variants and their population structure were assessed using median joining network and Bayesian approaches. While patterns of viral evolution clearly differed between subjects, immune system dysfunction related to HIV coinfection or persistent HCV seronegativity stand as potential mechanisms to explain the lack of molecular evolution observed in these three cases. In contrast, treatment of HCV infection with PegIFN, which did not lead to sustained virologic responses in all 3 cases, was not associated with commensurate variations in the complexity of the variant spectrum. Finally, the differences in the degree of divergence suggest that the mode of transmission of the virus was not the main factor driving viral evolution.

Molecular epidemiology of autochthonous dengue virus strains circulating in Mexico

ABSTRACT Dengue virus (DENV) is the most important arthropod-borne viral infection in humans. Here, the genetic relatedness among autochthonous DENV Mexican isolates was assessed. Phylogenetic and median-joining network analyses showed that viral strains recovered from different geographic locations are genetically related and relatively homogeneous, exhibiting limited nucleotide diversity.

Comparison between In Vitro Antiviral Effect of Mexican Propolis and Three Commercial Flavonoids against Canine Distemper Virus

Propolis is a resin that honey bees (Apis mellifera) produce by mixing wax, exudates collected from tree shoots, pollen, and enzymes. It has been used for its biological properties against pathogenic microorganisms including those of viral origin. In the present study, we demonstrate the antiviral effect of Mexican propolis, as well as of the three commercial flavonoids (quercetin, naringenin, and pinocembrin) present in its composition, in cell cultures infected with Canine Distemper Virus. The treatments were carried out with propolis, flavonoids individually, and a mixture of the three flavonoids at three different times. Antiviral activity was evaluated by the inhibition of the relative expression of the virus nucleoprotein gene (Real-Time qPCR) and by the determination of cellular viability (MTT assay). Propolis applied before infection decreased viral expression (0.72 versus 1.0, 1.65, and 1.75 relative expressions) and correlated with increased cell viability (0.314 versus 0.215, 0.259, and 0.237 absorbance units (AU)). The administration of a flavonoid mixture containing the three commercial flavonoids before infection induces a slight decrease in viral expression (0.93 versus 1, 1.42, and 1.82 relative expressions); however, it does not improve cellular viability (0.255 versus 0.247, 0.282, and 0.245 AU). Quercetin administrated at the same time of infection decreases viral expression (0.90 versus 1.0, 3.25, and 1.02 relative expressions) and improves cellular viability (0.294 versus 0.240, 0.250, and 0.245 AU). Pinocembrin and naringenin individually did not show any antiviral activity at the administration times evaluated in this study. The present work is the first in vitro study of the effect of propolis in Canine Distemper Virus and demonstrated the antiviral activity of Mexican propolis, in addition to the synergy that exists between the three flavonoids on cell viability and the expression of the nucleoprotein virus gene.