Karina Ruiz-Tovar

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.

Identification of Hepatitis C Virus transmission using a Next Generation Sequencing Approach

ABSTRACT Here, we describe a transmission event of hepatitis C virus (HCV) among injection drug users. Next-generation sequencing (NGS) was used to assess the intrahost viral genetic variation. Deep amplicon sequencing of HCV hypervariable region 1 allowed for a detailed analysis of the structure of the viral population. Establishment of the genetic relatedness between cases was accomplished by phylogenetic analysis. NGS is a powerful tool with applications in molecular epidemiology studies and outbreak investigations.

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.

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.

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.

Genetic relatedness among Japanese HAV isolates, 2010.

We have read with interest the recent article by Ishii et al.1 In the anuscript the authors describe the partial molecular characteriation of Japanese HAV isolates. While the information presented n the article is relevant, several issues are of concern. Here, we oint out some of the shortcomings and interpretation of the data er se. Firstly, “resurgence” might not be appropriate to refer to the scillating behavior of hepatitis A observed in Japan during the past ecade. As highlighted by the authors in their Fig. 2A, an increase n HAV cases was seen in 2010; however, a comparable number of ases were identified in 2006. While the number of cases seems to ave been reduced after year 2002, the fluctuation in the number f infections reported in more recent years might not be signifcant. Moreover, the PCR positivity rate among these specimens as 62.2%. Thus, more than 37% of the samples tested, presumably AV positive cases, were PCR negative. The positivity rates should ave been considerably higher if one assumes that anti-HAV IgM as used to identify the cases and that blood collection was perormed in a timely manner. Unfortunately, all these details are not entioned in the article and the reader is left to speculate on the ssue. Second, the term diffuse outbreak is used loosely. The artile does not present epidemiological information supporting the ssociation to a common source of infection. While some of these solates are most probably related, the possibility of some of these solates to be merely sporadic unrelated cases cannot be ruled out. Third, and more importantly, the conclusions derived from he partial molecular characterization and corresponding phyloenetic analyses do not seem to be supported. Several studies ave shown the limitations of using the VP1-2A junction to stablish relatedness.2,3 Therefore, partial genomic sequences will ever achieve the reliability of whole genome sequences. This is f particular importance when no epidemiological data is used o supplement the analysis. When strong epidemiological data s available, the robustness of the genetic relatedness study is trengthened.4–6 However, in the absence of such valuable inforation, complete homology of such small subgenomic region is not early enough to establish relatedness. It is actually quite common o find fairly different unrelated isolates sharing identical VP1-2A equences. Furthermore, a rather comprehensive study has clearly hown that unrelated viral strains sharing the same VP1-2A can