Phylogenetic and molecular characterization of coronavirus affecting species of bovine and birds in Cuba

REPORT Phylogenetic and molecular characterization of coronavirus affecting species of bovine and birds in Cuba Ana M Acevedo1, Nadia Martínez1, Paulo Brandão2, Carmen L Perera1, María T Frías1, Maritza Barrera1, " Lester J Pérez1 Grupo de Virología Animal, Dirección de Microbiología, Centro Nacional de Sanidad Agropecuaria, Censa AP 10, San José de las Lajas, Mayabeque, Cuba 2 Department of Preventive Veterinary Medicine and Animal Health, College of Veterinary Medicine, University of São Paulo, São Paulo, Brazil " 1 ABSTRACT Avian infectious bronchitis virus (IBV) and bovine coronavirus (BCoV) are pathogens of veterinary importance that affect birds and bovine in Cuba; however, molecular characteristics and genetic diversity of these viruses are unknown. This study was aimed at determining the molecular characteristics and genetic diversity of both agents, based in the spike S gene. A molecular analysis was carried out from field strains of BCoV collected between 2009 and 2011 and phylogenetic studies were conducted using partial or complete S gene sequences as phylogenetic markers. Besides, studies of phylogenetic inference were carried out in S1 region of recent isolates of IBV. All Cuban bovine coronavirus sequences were located in a single cluster supported by 100 % bootstrap and 1.00 posterior probability values. The Cuban BCoV sequences were also clustered with the USA BCoV strains corresponding to the GenBank accession numbers EF424621 and EF424623, suggesting a common origin for these viruses. This phylogenetic cluster was also the only group of sequences in which no recombination events were detected. Of the 45 amino acid changes found in the Cuban strains, four were unique. On the other hand, two putative genotypes genetically different to the Massachusetts genotype H120 strain used in the Cuban vaccination program were found in the flocks assessed. In addition, a potential nephropathogenic IBV isolate was found by first time in Cuba. This research won the 2012 Award of the Cuban National Academy of Sciences. Keywords: avian infectious bronchitis virus, bovine coronavirus, phylogenetic and molecular characterization Biotecnología Aplicada 2013;30:228-231 RESUMEN Caracterización filogenética y molecular de Coronavirus que afectan a especies de bovinos y aves en Cuba. El virus de la bronquitis infecciosa aviar (IBV) y el coronavirus bovino (BCoV) son agentes patógenos de importancia veterinaria porque afectan a las aves y al ganado bovino en Cuba. Como se desconocen sus características moleculares y diversidad genética, el objetivo de esta investigación fue determinarlas en ambos agentes, a partir del gen de la espícula S. Se analizaron cepas de campo de BCoV, recolectadas entre los años 2009 y 2011, y se compararon filogenéticamente según varios marcadores del gen S. Además se hicieron estudios de inferencia filogenética en una región de S1 de aislados recientes del IBV. Todas las cepas de BCoV cubanas se localizaron en un mismo grupo filogenético con un soporte estadístico del 100 % y valor de probabilidad posterior 1.00. Las secuencias de coronavirus bovino cubanas se agruparon con cepas de BCoV de EE.UU. con números de acceso en el GenBank de EF424621 y EF424623, que sugiere un origen común para estos virus. Este grupo filogenético fue el único en cuyas secuencias no se detectaron eventos de recombinación. De los 45 cambios de aminoácidos en las cepas cubanas, cuatro fueron únicos. En las granjas evaluadas se detectaron dos posibles genotipos genéticamente diferentes al genotipo Massachusetts de la cepa H120 usada en el programa de vacunación cubano. Además, se halló un potencial aislado de IBV nefropatogénico encontrado por primera vez en Cuba. Este trabajo mereció el Premio Anual de la Academia de Ciencias de Cuba del año 2012. Palabras clave: virus de la bronquitis infecciosa aviar, coronavirus bovino, caracterización filogenética y molecular Introduction The Coronavirus family has been studied for more than 50 years like virus that infect different species of animals, including the human. Several of these viruses, as the bovine coronavirus (BCoV) and infectious bronchitis virus (IBV), are of veterinary importance due to the respiratory and gastrointestinal disorders that they produce in domestic animals and the continuous emergency of new species [1], groups [2] or variants. In the case of IBV the continuous emergency and dissemination of different geno/serotypes have committed in the world the control of the disease caused " Corresponding author by this agent. Several reports have showed the continuous emergency of new variants caused by the evolution of field strains [3-4] or recombination events between heterologous strains classified in different genetic groups [5]. Therefore, identifying the diversity and molecular characteristics of the geno/serotypes that circulate in the populations is an indispensable task for controlling them. On the other hand, a better understanding of the molecular bases of BCoV evolution, tropism and virulence have been continuously demanded from the scientific community due to its economic relevance for cattle industry and its biological 1. Jackwood MW, Boynton TO, Hilt DA, McKinley ET, Kissinger JC, Paterson AH, et al. Emergence of a group 3 coronavirus through recombination. Virology. 2010;398(1):98-108. 2. Wood MK, Ladman BA, Preskenis A, Pope CR, Bautista DA, Gelb J Jr. Massachusetts Live Vaccination Protects Against a Novel Infectious Bronchitis Virus S1 Genotype DMV/5642/06. Avian Dis. 2009;53:119-23. Ana María Acevedo et al. Report and antigenic similarities with another Betacoronavirus as the human coronavirus OC43 (HCoV OC43). Additionally, the BCoV shares biological pathogenic and pneumoenteric properties with species of Coronavirus related with the SARS (SARS-CoVs) [6]. In order to fulfill its primary objectives on behalf of its mission to “Preserve the animal and human health”, the Center for animal Health (Censa, Cuba) required to incorporate all the genetic and molecular information on animal emergent pathogens. In this sense, it follows the purpose of developing diagnostic assays to detect the escape of new strains, tracing their origin and dissemination and to determine their potential virulence and pathogenicity. This research won the 2012 Award of the Cuban National Academy of Sciences. The objectives of present work were: To determine the genetic diversity and the phylogenetic relationships among infectious bronchitis virus (IBV) isolates associated with respiratory disease from Cuban chicken flocks based on partial S1 phylogenetic marker; and to compare the amino acidic identities of the spike gene vaccine virus and the isolates circulating in the poultry farms of Cuba. Additionally, regarding Bovine coronavirus (BCoV), the studies were intended to determine the genetic diversity and the phylogenetic relationships among different isolates of BCoV associated with similar diseases to WD and (...truncated)