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      Caracterización molecular del segmento 4 del virus de la tilapia de lago aislado de tilapias (Oreochromis niloticus) cultivadas en Perú Translated title: Molecular characterization of segment 4 of lake tilapia viruses isolated from tilapia (Oreochromis niloticus) farmed in Peru


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          RESUMEN El objetivo del presente estudio fue la caracterización molecular del segmento 4 del virus de la tilapia de lago (TiLV) detectado en tilapias de cultivo de los departamentos de Piura (Costa) y San Martín (Selva) en un brote ocurrido en 2017-2018. En el brote se obtuvo 26 muestras positivas a TiLV, de las cuales se seleccionaron cinco muestras positivas. El diagnóstico de estas muestras se realizó a través de un RT-PCR anidada con cebadores dirigidos al segmento 3 y los productos de PCR fueron secuenciados. Para la amplificación y análisis del segmento 4 del genoma del TiLV se realizó un RT-PCR donde se diseñaron cebadores específicos. La secuenciación se hizo con la empresa Macrogen (Corea del Sur), mediante secuenciación bidireccional por el método de Sanger automatizado. El análisis filogenético se realizó a partir de las secuencias alineadas por medio del método de Neighbor-Joining (NJ) y la característica de la proteína hipotética del gen se realizó con el programa Phyre2. Se obtuvieron cuatro secuencias completas del segmento 4 (1 de Piura y 3 de San Martín) con una longitud de 1190 pb siendo comparadas con dos secuencias de Israel y una de Tailandia, cepas referenciales correspondientes al segmento 4 de TILV publicadas GeneBank. El análisis filogenético del segmento 4 determinó la presencia de un genogrupo TiLV local, además de indicar que las muestras peruanas presentan mayor relación genética con el clado de cepas de Israel. El análisis de distancia genética muestra que las muestras peruanas mostraron valores de identidad nucleotídica de 99.7-100% entre ellas, determinando que los brotes de ambos lugares fueron producidos por la misma cepa viral, y tienen una identidad de 97.5-97.7% con cepas de Israel y 97.0-97.1% con cepas de Tailandia. Se determinó que la proteína hipotética a partir del segmento 4 del TiLV tiene una región con una homología estructural con la proteína neuraminidasa N6 del pato inglés con 12% de cobertura, 44% de identidad y 24.9% de confianza.

          Translated abstract

          ABSTRACT The aim of this study was the molecular characterization of segment 4 of the lake tilapia virus (TiLV) detected in farmed tilapia from the departments of Piura (Coast) and San Martín (Jungle) in an outbreak that occurred in 2017-2018. During this outbreak, 26 TiLV positive samples were obtained and five of them were selected. The diagnosis of these samples was carried out through a nested RT-PCR with primers directed to segment 3 and the PCR products were sequenced. For the amplification and analysis of segment 4 of the TiLV genome, an RT-PCR was performed where specific primers were designed. The sequencing was done by Macrogen (South Korea), by bidirectional sequencing using the automated Sanger method. The phylogenetic analysis was carried out from the aligned sequences by means of the Neighbor-Joining (NJ) method and the hypothetical protein characteristics of the gene was carried out with the Phyre2 program. Four sequences with a length of 1190 bp were obtained and compared with two sequences from Israel and one from Thailand, reference strains corresponding to segment 4 of TILV published GenBank. The phylogenetic analysis of segment 4 determined the presence of a local TiLV genogroup, in addition to indicating that the Peruvian samples have a greater genetic relationship with the clade of Israel strains. The genetic distance analysis shows that the Peruvian samples have nucleotide identity values of 99.7-100% between them, determining that the outbreaks of both locations were produced by the same viral strain, and have an identity of 97.5-97.7% with strains from Israel and 97.0-97.1% with strains from Thailand. The hypothetical protein from TiLV segment 4 was determined to have structural homology to the neuraminidase N6 protein from English duck influenza A virus with 12% coverage, 44% identity, and 24.9% confidence.

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          Most cited references32

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          MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets.

          We present the latest version of the Molecular Evolutionary Genetics Analysis (Mega) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, Mega has been optimized for use on 64-bit computing systems for analyzing larger datasets. Researchers can now explore and analyze tens of thousands of sequences in Mega The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit Mega is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OS X. The command line Mega is available as native applications for Windows, Linux, and Mac OS X. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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            The Phyre2 web portal for protein modeling, prediction and analysis.

            Phyre2 is a suite of tools available on the web to predict and analyze protein structure, function and mutations. The focus of Phyre2 is to provide biologists with a simple and intuitive interface to state-of-the-art protein bioinformatics tools. Phyre2 replaces Phyre, the original version of the server for which we previously published a paper in Nature Protocols. In this updated protocol, we describe Phyre2, which uses advanced remote homology detection methods to build 3D models, predict ligand binding sites and analyze the effect of amino acid variants (e.g., nonsynonymous SNPs (nsSNPs)) for a user's protein sequence. Users are guided through results by a simple interface at a level of detail they determine. This protocol will guide users from submitting a protein sequence to interpreting the secondary and tertiary structure of their models, their domain composition and model quality. A range of additional available tools is described to find a protein structure in a genome, to submit large number of sequences at once and to automatically run weekly searches for proteins that are difficult to model. The server is available at http://www.sbg.bio.ic.ac.uk/phyre2. A typical structure prediction will be returned between 30 min and 2 h after submission.
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              A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences.

              Some simple formulae were obtained which enable us to estimate evolutionary distances in terms of the number of nucleotide substitutions (and, also, the evolutionary rates when the divergence times are known). In comparing a pair of nucleotide sequences, we distinguish two types of differences; if homologous sites are occupied by different nucleotide bases but both are purines or both pyrimidines, the difference is called type I (or "transition" type), while, if one of the two is a purine and the other is a pyrimidine, the difference is called type II (or "transversion" type). Letting P and Q be respectively the fractions of nucleotide sites showing type I and type II differences between two sequences compared, then the evolutionary distance per site is K = -(1/2) ln [(1-2P-Q) square root of 1-2Q]. The evolutionary rate per year is then given by k = K/(2T), where T is the time since the divergence of the two sequences. If only the third codon positions are compared, the synonymous component of the evolutionary base substitutions per site is estimated by K'S = -(1/2) ln (1-2P-Q). Also, formulae for standard errors were obtained. Some examples were worked out using reported globin sequences to show that synonymous substitutions occur at much higher rates than amino acid-altering substitutions in evolution.

                Author and article information

                Revista de Investigaciones Veterinarias del Perú
                Rev. investig. vet. Perú
                Universidad Nacional Mayor de San Marcos. Facultad de Medicina Veterinaria (Lima, , Peru )
                April 2021
                : 32
                : 2
                [2] Lima Lima orgnameUniversidad Nacional Mayor de San Marcos orgdiv1Facultad de Medicina Veterinaria orgdiv2Laboratorio de Microbiología y Parasitología Veterinaria Peru
                [1] Lima Lima orgnameUniversidad Nacional Mayor de San Marcos orgdiv1Facultad de Medicina Veterinaria orgdiv2Laboratorio de Histología, Embriología y Patología Veterinaria Peru nsandovalc@ 123456unmsm.edu.pe
                S1609-91172021000200012 S1609-9117(21)03200200012

                This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

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                tilapias,tilapia,TiLV,segment 4,RT-PCR,análisis filogenético,segmento 4,phylogenetic analysis


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