2. Justificativa
4.5. Análise filogenética e construção de Median
Apenas fragmentos cobrindo todo o comprimento do amplificon, incluindo os MIDs em ambas as extremidades, foram analisados. O alinhamento múltiplo foi realizado com o programa MAFFT v7 (Katoh and Standley 2013). Os alinhamentos otimizados foram então analisados em MEGA 5 e árvore filogenéticas construídas com o método de Neighbor-
Joining (Tamura, Peterson et al. 2011). A variação genética intra-
hospedeiro também foi analisada utilizando a análise de Median Joining
Networks (Bandelt, Forster et al. 1999), tal como implementada em
Network v4.6 (Fluxus Technology) como descrito anteriormente (Escobar- Gutierrez, Soudeyns et al. 2013).
35
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41
Capítulo 2
Research paper
Multiregion deep sequencing of hepatitis C virus: An improved approach
for genetic relatedness studies
Livia Maria Gonçalves Rossia,b,⁎, Alejandro Escobar-Gutierrezb, Paula Rahala
aDepartment of Biology, Institute of Bioscience, Language and Exact Science, São Paulo State University, São José do Rio Preto, Sao Paulo, Brazil bInstituto de Diagnóstico y Referencia Epidemiológicos, Mexico City, Mexico
a b s t r a c t a r t i c l e i n f o
Article history:
Received 22 November 2015
Received in revised form 23 December 2015 Accepted 24 December 2015
Available online 28 December 2015
Hepatitis C virus (HCV) is a major public health problem that affects more than 180 million people worldwide. Identification of HCV transmission networks is of critical importance for disease control. HCV related cases are often difficult to identify due to the characteristic long incubation period and lack of symptoms during the acute phase of the disease, making it challenging to link related cases to a common source of infection. Addition- ally, HCV transmission chains are difficult to trace back since viral variants from epidemiologically linked cases are genetically related but rarely identical. Genetic relatedness studies primarily rely on information obtained from the rapidly evolving HCV hypervariable region 1 (HVR1). However, in some instances, the rapid divergence of this region can lead to loss of genetic links between related isolates, which represents an important challenge for outbreak investigations and genetic relatedness studies. Sequencing of multiple and longer sub-genomic re- gions has been proposed as an alternative to overcome the limitations imposed by the rapid molecular evolution of the HCV HVR1. Additionally, conventional molecular approaches required to characterize the HCV intra-host genetic variation are laborious, time-consuming, and expensive while providing limited information about the composition of the viral population. Next generation sequencing (NGS) approaches enormously facilitate the characterization of the HCV intra-host population by detecting rare variants at much lower frequencies. Thus, NGS approaches using multiple sub-genomic regions should improve the characterization of the HCV intra- host population. Here, we explore the usefulness of multiregion sequencing using a NGS platform for genetic relatedness studies among HCV cases.
© 2015 Elsevier B.V. All rights reserved. Keywords:
Hepatitis C virus Next generation sequencing Outbreak
Multiregion Genetic relatedness
1. Introduction
Globally, hepatitis C virus (HCV) affects more than 180 million people (Mohd Hanafiah et al. 2013), in addition to 3–4 million new infections per year (Alter 2007; Lavanchy 2009). HCV infection is one of the leading causes of chronic liver disease associated with end- stage cirrhosis and hepatocellular carcinoma (Lauer and Walker 2001; McHutchison and Bacon 2005).
HCV is a small single-stranded, positive polarity, enveloped virus be- longing to the Hepacivirus genus within the Flaviviridae family (Smith et al. 2014). The RNA viral genome (~9.6 kb in length) contains a single open reading frame encoding for a long polyprotein that upon matura- tion by enzymatic cleavage originates three structural proteins and seven non-structural proteins (Chevaliez and Pawlotsky 2006; Stanley et al. 2007). The HCV RNA replication process is highly error prone (Moradpour et al. 2007), and so far seven major HCV genotypes and several sub-types have been identified (Smith et al. 2014). Introduction of point mutations by the RNA polymerase is the primary element
contributing to the high genetic variability of HCV. The HCV mutation rate in vivo is ~ 2.5 × 10−5per nucleotide per genome replication (Ribeiro et al. 2012); however, higher estimates have also been reported (Cuevas et al. 2009).
Rapid detection of HCV outbreaks and implementation of proper disease control measures are crucial to prevent virus spread and provide adequate health care. However, HCV transmission networks are difficult to identify. The intricate patterns of HCV molecular evolu- tion and silent onset of disease complicate the recognition of transmis- sion events (Goncalves Rossi and Rahal 2014). The hypervariable region 1 (HVR1) is generally used to characterize the HCV intra-host population (Campo et al. 2014; Forbi et al. 2014), and to detect HCV transmission by assessing the genetic relatedness of HVR1 variants among infected patients (Campo et al. 2015; Cruz-Rivera et al. 2013; Escobar-Gutierrez et al. 2013; Escobar-Gutierrez et al. 2012; Gismondi et al. 2013; Rossi et al. 2015). However, there are intrinsic limitations imposed by the use of a rapidly evolving sub-genomic region (Preciado et al. 2014; Rossi et al. 2015). Over time, genetic links can be lost due to rapid sequence divergence, impairing outbreak investigation studies (Cruz-Rivera et al. 2013). Thus, we propose that sequencing of additional sub-genomic regions might aid to restore links between
Infection, Genetics and Evolution 38 (2016) 138–145
⁎ Corresponding author.
E-mail address:liv.rossi@yahoo.com(L.M. Gonçalves Rossi).
http://dx.doi.org/10.1016/j.meegid.2015.12.020
1567-1348/© 2015 Elsevier B.V. All rights reserved.
Contents lists available atScienceDirect
Infection, Genetics and Evolution
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / m e e g i disolates. Here, deep sequencing of the HCV HVR1 supplemented with sequences originated from the NS5A region was used to establish relat- edness among HCV cases.
2. Materials and methods 2.1. Clinical samples
HCV chronic cases, aged 34–58 years, were enrolled in this study. These patients were a sub-set of a larger cohort (Cruz-Rivera et al. 2013; Escobar-Gutierrez et al. 2012; Fonseca-Coronado et al. 2012). All patients were anti-HCV treatment-naïve reporting injection drug users (IDU) activity. Ethical review and informed consent approval were granted by the Ethical Committee of the reference laboratory in Mexico. Informed consent was obtained from all subjects. Plasma samples from all subjects were obtained and stored at − 70 °C until use. Patients' characteristics are summarized inTable 1.
2.2. Amplicon deep sequencing
The intra-host viral genetic variation in each patient was evaluated