viernes, 9 de febrero de 2018

HITOS DE LA INFLUENZA EQUINA EN CHILE. P. Berríos E. 2018

Hitos de la influenza equina (IE) en Chile ¿Endémica, cíclica?
Patricio Berríos E.
2018

1) En junio de 1963 se describió el primer brote de IE en Chile. Según Fuschlocher en 1957 se habría presentado un cuadro similar, incluso recordaba que en 1936 había ocurrido una enfermedad semejante (Fuschlocher et al, 1963).
2) En otoño de 1973 se presentó IE en un brote de larga duración (Ríos, 1973).
3) En enero de 1977 se presentó IE de inusitada intensidad causando cuantiosas pérdidas económicas. El virus causante fue el H7 N7 (Casanova et al, 1977; Muñoz et al, 1979).
4) En diciembre de 1985 se describe IE con una tasa de ataque alta. El virus causante fue el H3 N8 (Berríos et al, 1986).
5) En enero de 1992 se presentó IE más suave causado por el H3 N8.
6) En junio de 2006 se describe IE causada por el H3 N8 (Méndez et al, 2006).
7) En enero de 2012 se inició en Los Andes un brote de enfermedad respiratoria causado por el H3 N8.
8) En 2013 se presentaron casos en Magallanes en que el SAG confirmó la presencia del virus IE.
9) En enero de 2018 se determina la presencia de IE causada por el H3 N8. Brote en curso...

¿Es endémica la IE en Chile? ¿Se presenta siguiendo ciertos ciclos epidemiológicos? ¿Dónde se inició el brote 2018?

viernes, 19 de enero de 2018

Especial verde y sustentable: Términos que cuentan 2018

Agujero en la capa de ozono
Se refiere a la pérdida periódica de ozono -cuya función es la protección contra las radiaciones ultravioletas emitidas por el Sol- en las capas superiores de la atmósfera en la zona de la Antártida. El agujero, provocado por los clorofluorocarbonos (CFC) y otros elementos químicos usados por mucho tiempo como refrigerantes, se presenta durante la primavera antártica y dura varios meses antes de cerrarse de nuevo. Gracias a las progresivas restricciones a los CFC, la NASA cree que el agujero podría desaparecer en 2065.

B
Biodegradable
Sustancia que puede descomponerse a través de procesos biológicos realizados por diversos microorganismos. La biodegrabilidad de los materiales depende de su estructura física y química, por lo que el plástico tradicional es menos propenso a este proceso que el papel. Por ejemplo, algunas bolsas biodegradables que se usan en el retail nacional se desintegran por completo en 15 meses, mientras que las de plástico tradicional demoran hasta 300 años. Mucho del plástico que se utiliza va a dar al mar, lo que ha generado una crisis en los océanos, razón por la cual a comienzos de este mes en Chile se aprobó un proyecto que busca prohibir gradualmente la entrega de bolsas plásticas por parte del comercio, primero en las 102 comunas costeras y luego para que en las demás se establezca una ordenanza para regular su uso.
Biodiversidad
Es la variedad de plantas, animales, hongos y microorganismos que viven en un espacio determinado, además de su variabilidad genética y los paisajes o regiones en donde se ubican. Los países con mayor biodiversidad del mundo son Brasil, Colombia, Indonesia, China, México, Perú, Australia, India, Ecuador y Venezuela.
Biomasa
Fresh and mature home compost piles.
Materia orgánica de origen vegetal o animal, incluyendo los residuos y desechos orgánicos, susceptible de ser aprovechada energéticamente. Es el caso del biogás, que se produce cuando se pudren elementos de la basura y sirve para tareas como la calefacción. Otro ejemplo es el de la Región de Biobío, donde ya hay 13 plantas de biomasa que procesan desechos forestales como astillas, y generan energía que aportan al sistema interconectado central.
C
Cambio climático
Alteraciones de los ciclos naturales del planeta por efecto de la actividad humana, especialmente las emisiones masivas de dióxido de carbono (CO2) a la atmósfera provocadas por las industrias y el uso masivo de combustibles fósiles. Según cifras de 2017, los niveles en la atmósfera de CO2 –gas que atrapa el calor- superan en 46 por ciento a los existentes a inicios de la revolución industrial.
Calentamiento global
Es el aumento de la temperatura del planeta producto de la actividad humana en los últimos 100 años, lo que modifica las estaciones de lluvia y aumenta el nivel del mar. Durante el pasado siglo, se calcula que la temperatura media global ha aumentado 0,76 °C, mientras que en zonas como el Ártico ese índice llega hasta los 5 °C.
Combustibles fósiles
Son aquellos que proceden de la biomasa producida hace millones de años y que pasaron por procesos de transformación hasta generar sustancias de gran contenido energético y no renovables, como el carbón y el petróleo. Estos combustibles liberan compuestos como CO2 y conformarán el 70 por ciento del consumo mundial de energía hasta el año 2040.
Garbage lie at Dbayeh's seaside shore north of Beirut on November 28, 2017 after it was washed away by the sea from the nearby seaside garbage dump of Karanatina. The open burning of waste in Lebanon poses serious health risks, Human Rights Watch warned in a report released today, blaming decades-old, across the board government failure. The New York-based watchdog said the crisis, which escalated in 2015 when waste management largely collapsed across Lebanon, was a particular threat for children and old people, and constituted a rights violation. / AFP PHOTO / JOSEPH EID LEBANON-ENVIRONMENT-WASTE
Contaminación
El término proviene del latín “contaminare” (manchar) y alude a la introducción de sustancias u otros elementos físicos en un medio que provocan un cambio perjudicial en las características químicas, físicas y biológicas de un ambiente o entorno. Esta alteración, que puede ser provocada por factores como las sustancias químicas del esmog, un sonido o radiactividad, entre otros, afecta o puede incidir en la vida de organismos y humanos. Actualmente, la ciudad con el aire más contaminado del mundo es Zabol, en Irán.
Consumo responsable
También conocido como “consumo justo”, consiste en elegir productos no sólo en base a su calidad y precio, sino también por su impacto ambiental y social, y por los procesos de producción, transporte y distribución que realizan las empresas que los elaboran. La idea es cambiar los hábitos de compra ajustándolos a las necesidades del planeta y escogiendo opciones que favorezcan el medioambiente y la igualdad social.
Cumbre ODS
Se realizó en septiembre de 2015 en Nueva York y en ella los estados miembros de la ONU aprobaron la Agenda 2030 para el Desarrollo Sostenible, que incluye 17 Objetivos de Desarrollo Sostenible (ODS) para poner fin a la pobreza, luchar contra la desigualdad y la injusticia, y hacer frente al cambio climático. Entre los fines está gestionar los bosques de forma sostenible, luchar contra la desertificación, resguardar los recursos marinos y lograr que las ciudades y los asentamientos humanos sean inclusivos, seguros, resilientes y sostenibles.
D
Deforestación
Desaparición o disminución de las superficies cubiertas por bosques, producto de labores madereras, uso de los árboles como combustible y la creciente extensión de superficies destinadas a cultivos y pastoreo. Según estudios internacionales, el bosque nativo chileno de la zona central ha perdido el 83 por ciento de su vegetación original.
Desertificación
Proceso por el cual un territorio que no posee las condiciones climáticas de un desierto adquiere sus características, como resultado de la destrucción de su cubierta vegetal y de la fuerte erosión. La sobreexplotación de los suelos, el abuso de pesticidas y plaguicidas, la tala indiscriminada de árboles y el cambio climático favorecen este fenómeno. Por ejemplo, Conaf estima que en 2050 la zona de Coquimbo tendría las condiciones de aridez de Atacama.
E
Ecología
Ciencia que estudia a los seres vivos en sus distintos niveles de organización y sus interrelaciones entre ellos y con el medioambiente. El término fue acuñado en 1869 por el naturalista y filósofo alemán Ernst Haenckel, quien unió las palabras griegas oikos (casa, hogar) y logos (estudio).
Ecosistema
Complejo dinámico de comunidades vegetales, animales microorganismos y su entorno. La zona de la Amazonía se considera como el ecosistema más rico del mundo: sólo en el río que recorre el sector conviven más de 2.500 especies de peces.
Educación ambiental
Proceso de formación que permite la toma de conciencia de la importancia del medioambiente, promueve en la ciudadanía el desarrollo de valores y actitudes que contribuyan al uso racional de los recursos naturales y a la solución de los problemas ambientales. Desde 2003, más de 1.300 establecimientos de nivel parvulario, básico y medio en Chile han iniciado procesos de certificación ambiental que incluyen currículo y metodologías de educación ambiental.
19 de julio 2017/SANTIAGO Inauguran la planta de energía solar fotovoltaica, mas grande la Región Metropolitana llamada Quilapilún, el evento contó con la presencia del ministro de Energía, Andrés Rebolledo y el de Medio Ambiente, Marcelo Mena FOTO: FRANCISCO CASTILLO D./AGENCIAUNO
Energías alternativas
También llamadas renovables. Se renuevan siempre y se refieren, por ejemplo, a la energía solar, la eólica, la hidráulica, la biomasa o la geotérmica (calor natural de la Tierra). En Chile la energía solar ya genera el siete por ciento de la electricidad del país y representa el 44 por ciento de la producción de energías limpias.
G
Gases de invernadero
Gases como el dióxido de carbono o el metano que se acumulan en la atmósfera y actúan como un techo que limita el ritmo de escape del calor de Sol desde la superficie terrestre. En el caso del metano, proviene de fuentes como la digestión que realiza el ganado: un estudio divulgado en 2017 por la NASA señala que sólo en 2011 vacas y otros animales liberaron 119 millones de toneladas de ese gas.
H
23 de Septiembre del 2013/PAINE Una garza camina por un humedal, durante este segundo día de primavera. Oficialmente esta estación comenzó el día de ayer, al mediodía, cuando se produjo el "Equinoccio de primavera", momento en que el sol pasa desde el hemisferio norte hacía el hemisferio sur, teniéndo desde entonces, el día, la misma duración que la noche. FOTO: HANS SCOTT/AGENCIAUNO.
Humedal
Se refiere a una amplia variedad de ambientes que comparten la presencia del agua como elemento característico. Según la Convención sobre los Humedales (Ramsar, Irán, 1971), estas áreas abarcan marismas, pantanos, turberas y extensiones de agua marina que regulan procesos hidrológicos y operan sobre todo como hábitats de aves acuáticas. Chile tiene 13 sitios designados como “Humedales de Importancia Internacional”, con una superficie de 361,761 hectáreas.
Huella de carbono
Es la totalidad de gases de efecto invernadero emitidos por efecto directo o indirecto de un individuo, organización, evento o producto. Por ejemplo, gracias a la popularidad de serviciosstreaming como YouTube, Netflix y Spotify la industria del entretenimiento ha reducido 40 por ciento sus emisiones de dióxido de carbono, que venían de la elaboración de CD, DVD, el celofán y las cajas de acrílico o plástico necesarias para empacar los discos.
I
Impacto ecológico
Abarca los efectos producidos por las actividades humanas en organismos vivientes y en su medioambiente no viviente. Estos incluyen, por ejemplo, desde la contaminación del mar con petróleo, los desechos radiactivos y la emisión de gases nocivos.
L
Lluvia ácida
Fenómeno contaminante que se produce al combinarse el vapor de agua atmosférico con óxidos de azufre y de nitrógeno, formando ácido sulfúrico y ácido nítrico. Esto cae como precipitación que deteriora los lagos, los árboles y otras especies y se origina sobre zonas donde se queman combustibles fósiles. Uno de los países donde este fenómeno empeora día a día es la India, a raíz del alto uso del carbón. 25 Marzo de 2015/ HUALPEN Vecinos de la comuna de Hualpén denunciaron nuevo episodio de malos olores emanados desde Enap, en donde la alcaldesa de Hualpén, Fabiola Lagos, llegó hasta la Corte de Apelaciones de Concepción para presentar un recurso de protección y que incluye una serie de mejoras al interior de la planta. La última emanación se produjo la semana pasada, donde incluso los vecinos afectados debieron acudir a recintos de salud. Enap se comprometió con más de US$13 millones para mitigar los malos olores de refinería de Hualpén FOTO: MARIBEL FORNEROD/AGENCIAUNO
N
Neutro en carbono
Se refiere a empresas u organizaciones con cero emisiones de dióxido de carbono. El nivel cero se puede conseguir mediante la reducción progresiva de emisiones, el uso de energías renovables o a partir de una combinación de ambas opciones. En Chile hay varias instituciones que son neutras en carbono, como el festival Lollapalooza, DHL y CorpBanca.
O
Orgánico
Se refiere a los productos agrícolas elaborados sin el uso de fertilizantes, pesticidas u otros químicos artificiales. De acuerdo con cifras internacionales, en la última década el mercado global de alimentos orgánicos aumentó en un 170 por ciento. En Chile hay 131.973 hectáreas certificadas como orgánicas, de las cuales el seis por ciento corresponde a frutales como la uva vinífera, los arándanos y los manzanos.
P
Parques naturales
Áreas poco transformadas por la explotación u ocupación humana que debido a la belleza de sus paisajes, la representatividad de sus ecosistemas o la singularidad de su flora, de su fauna o de sus formaciones geomorfológicas, poseen valor ecológico, estético, educativo o científico. Chile tiene 36 parques nacionales y dos parques marinos.
R
Las tres R
Reducir. Reutilizar. Reciclar. Es una propuesta de consumo, popularizada por Greenpeace, que apunta a desarrollar hábitos como el consumo responsable y busca reducir el consumo de bienes o energía, reutilizar para extender la vida útil de productos como el papel y reciclar materiales como el plástico. En 2004, el primer ministro de Japón, Koizumi Junichiro, presentó una iniciativa en la cumbre del G8 para promover globalmente una sociedad orientada hacia las tres R.
S
Sostenible
Crecimiento regulado que a través de medidas políticas y sociales busca utilizar de manera eficiente los recursos. Este tipo de desarrollo satisface las necesidades actuales de todos los habitantes del planeta, sin comprometer los recursos del futuro. Un ejemplo de desarrollo sostenible son las energías renovables.
Sustentable
Desarrollo que puede mantenerse por sí mismo sin que se vean afectados los recursos del planeta. No precisa una intervención humana o exterior, ya que puede sostenerse de manera autónoma. Por ejemplo, la Viña San Pedro utiliza estanques con biodigestores con microorganismos que procesan palos, pellejos y pepas de uva y generan gas que les permite abastecer en promedio el 60 por ciento de la energía eléctrica y térmica que requieren.
T
BY5BGR Corn cobs drying - Hungary
Transgénico
Producto vegetal que ha sido manipulado genéticamente con el objeto de mejorar su rendimiento productivo y, por lo tanto, la rentabilidad de su explotación. Aún se desconocen los efectos que estas alteraciones genéticas podrían provocar en otras especies, entre ellas la humana. En Chile, las principales semillas transgénicas producidas son el maíz, la canola y la soja.
V
Verde
Término que se aplica ampliamente para describir algún sitio diseñado de una manera respetuosa con el ambiente o para tener un impacto mínimo en él. Por ejemplo, el edificio The Edge en Holanda cuenta con 28 mil sensores para detectar humedad, temperatura y otros factores que le permiten ajustar su iluminación y el aire acondicionado.
W
World Wide Fund for Nature (WWF)
Fondo Mundial para la Naturaleza. Organización creada en 1961 en Suiza para ayudar a conservar la naturaleza y los procesos ecológicos esenciales para la vida en el planeta.
Z
Zero Waste
Significa “basura cero” y es una tendencia mundial que comenzó en los 90 y que busca reducir al mínimo la producción de residuos. Un ejemplo es el restaurante Panatura Vegano –ubicado a pasos de Plaza Ñuñoa–, que se encarga de sus residuos orgánicos, no entregan plástico y reutilizan las aguas grises de los baños

viernes, 17 de noviembre de 2017

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome Carlos A. Loncoman,a Carol A. Hartley,a Mauricio J. C. Coppo,a Paola K. Vaz,a Andrés Diaz-Méndez,a Glenn F. Browning,a Maricarmen García,b Stephen Spatz,c Joanne M. Devlina 2017

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome Carlos
A. Loncoman,a Carol A. Hartley,a Mauricio J. C. Coppo,a Paola K. Vaz,a Andrés Diaz-Méndez,a Glenn F. Browning,a Maricarmen García,b Stephen Spatz,c Joanne M. Devlina
Asia-Pacific Centre for Animal Health, Melbourne Veterinary School, Faculty of Veterinary and Agricultural Sciences, University of Melbourne, Parkville, Victoria, Australiaa ; Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USAb; Southeast Poultry Research Laboratory, Agricultural Research Service, United States Department of Agriculture, Athens, Georgia, USAc

ABSTRACT Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.

IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

KEYWORDS herpesvirus, recombination, replication, diversity, SNP genotyping assay, vaccine, infectious laryngotracheitis virus (ILTV), recombination hot spot, gallid herpesvirus 1, ILTV Received 12 July 2017 Accepted 12 September 2017 Accepted manuscript posted online 22 September 2017 Citation Loncoman, CA, Hartley CA, Coppo MJC, Vaz PK, Diaz-Méndez A, Browning GF, García M, Spatz S, Devlin JM. 2017. Genetic diversity of infectious laryngotracheitis virus during in vivo coinfection parallels viral replication and arises from recombination hot spots within the genome. Appl Environ Microbiol 83:e01532-17. https://doi.org/10 .1128/AEM.01532-17. Editor Harold L. Drake, University of Bayreuth Copyright © 2017 American Society for Microbiology. All Rights Reserved. Address correspondence to Carlos A. Loncoman, cloncoman@student.unimelb.edu.au. MICROBIAL ECOLOGY crossm December 2017 Volume 83 Issue 23 e01532-17 Applied and Environmental Microbiology aem.asm.org 1 on November 16, 2017 by guest http://aem.asm.org/

Downloaded from I nfectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) is an alphaherpesvirus that causes mild to severe respiratory disease in chickens. The virus causes major economic losses due to mortality and decreases in weight gain and egg production in poultry industries worldwide (1). Recombination between different strains of ILTV has been recognized as a problem for the poultry industry, particularly because natural recombination between attenuated vaccine strains of ILTV has been shown to generate virulent viruses (2). The importance of recombination as a mechanism of viral genome diversification and evolution in ILTV and other alphaherpesviruses is increasingly being recognized (3). Viruses can acquire genetic changes through several mechanisms, including point mutation and recombination, with the latter particularly important in many alphaherpesviruses (4–6). Viruses that belong to the order Herpesvirales have double-stranded linear DNA genomes and have complex viral DNA replication machinery, comprising a DNA polymerase with a highly efficient proofreading capacity, resulting in very low rates of spontaneous mutation (7–9). Therefore, rather than random changes or point mutations throughout the genome, recombination is considered to be a major evolutionary driving force enabling many alphaherpesviruses to persist, evolve and eventually become more virulent over time (2, 10, 11). Four types of recombination have been described, based on the structure of the crossover site: site-specific recombination, transposition recombination, nonhomologous or illegitimate recombination, and homologous recombination (12). Homologous and illegitimate recombination, specifically intraspecific recombination, are the most commonly described recombination mechanisms in alphaherpesviruses (12). Intraspecific recombination under in vitro conditions has been extensively described for several alphaherpesvirus species, including Human alphaherpesvirus 1, also known as herpes simplex virus 1 (HSV-1) (13), Bovine alphaherpesvirus 1, also known as bovine herpesvirus 1 (BoHV-1) (14), Human alphaherpesvirus 3, also known as varicella-zoster virus (VZV) (15), Felid alphaherpesvirus 1, also known as felid herpesvirus 1 (FeHV-1) (16) and Suid alphaherpesvirus 1, also known as pseudorabies virus (PRV) (17). Full-genome sequencing and sequence analysis are the most suitable methods for detecting and describing alphaherpesvirus recombination events that occur under natural (field) conditions, but even nowadays these techniques have their limitations, including intensive labor and costs. In contrast, under experimental conditions, other techniques to detect recombination are still more efficient and cost-effective, as well as more suitable for testing large numbers of viruses (14, 18). We have recently described the development and validation of a TaqMan single nucleotide polymorphism (SNP) genotyping assay to study recombination under experimental conditions in specific-pathogen-free (SPF) chickens (18), the natural host of ILTV. Use of the natural host to study recombination may reveal aspects of herpesvirus biology that may not be apparent in studies that utilize laboratory animal models of infection (19). The aim of this study was to apply this ILTV SNP genotyping assay to examine in vivo recombination between two ILTV strains over time. This was performed in order to describe recombination patterns and to examine genetic diversity among the viral progeny during the course of an ILTV infection. Additionally, we aimed to identify any recombination hot spots in the ILTV genome by performing full-genome sequencing of selected recombinants and analyzing them along with other ILTV genomes that were already available from different geographical regions, including Asia, Australia, Europe, and the United States. RESULTS Bird survival rates and virus genome quantification. Groups of chickens were coinoculated with the V1-99 and CSW-1 strains of ILTV at two different doses or were inoculated with only either V1-99 or CSW-1. The survival rates in groups of birds that were inoculated only with low or high doses of V1-99 ILTV were around 70% and 45% throughout the experiment, respectively (Fig. 1). Groups that received only CSW-1 had 100% survival rates throughout the experiment (Fig. 1). In the coinoculated groups, the Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 2 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from FIG 2 Replication of ILTV in SPF chickens based on genome copy numbers in tracheal swabs measured using qPCR. Medians are indicated by lines for each group. Birds were inoculated with either CSW-1 or V1-99 or coinoculated (co-inoc) with 103 PFU (low dose) (A to D) or with 104 PFU (high dose) (E to H) of the V1-99 and (Continued on next page) Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 4 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from lated with the low dose of viruses, 14, 26, 15, and 9 genotype patterns were detected at 2, 4, 6, and 8 days postinoculation, respectively (Tables 2 and 3). Some of these genotype patterns were detected consistently over time in both low- and high-doseinoculated groups. In the singly inoculated groups, no recombination was detected within the 10 viruses that were isolated and purified from each infected chicken at day 4 from the groups inoculated with the higher viral dose of 1 104 PFU/ml (see Fig. S1 in the supplemental material). Viral-diversity analysis. The higher level of viral replication in the low-dosecoinoculated group at day 4 than day 2 (Fig. 2A and B) coincided with a higher level of viral diversity, as assessed using Renyi diversity profiles (Fig. 5A) and as indicated by significantly higher values for diversity as measured by richness (0), ShannonWeaver (1), 1/Simpson (2), 1/Berger Parker (infinite [Inf]), and Hill (0.25, 0.5, 4, 8, 16, 32, and 64) scales, as well as evenness values at these time points (see Table S1 in the supplemental material). The Renyi diversity profile was higher at day 6 than at day 8 (Fig. 5B); however, the diversity measurements were not significantly different between these days (see Table S2). In the high-dose-coinoculated group, the Renyi diversity profile was higher at day 4 than at day 2 (Fig. 5C), coinciding with the higher levels of viral replication at day 4 than day 2 in this group (Fig. 2E and F). Significant differences were detected only in the Shannon-Weaver (1), 1/Simpson (2), and Hill (4) values for these days (see Table S3). Full-genome sequencing and identification of recombination hot spots. Six progeny viruses were selected for full-genome sequencing and analysis (see Table S4) in order to identify all recombination events and compare these to those detected with the SNP genotyping assay and to examine the distribution of recombination breakpoints along the genome. These recombination patterns were also searched for among 35 other full ILTV genome sequences of isolates from Asia, Australia, Europe, and the United States in order to identify recombination hot spots in ILTV from distinct geographical regions. In addition to the distribution of recombination breakpoints detected by RDP4, reticulate network and phi test analyses were performed as previously described (20) (see Fig. S2). The six SNPs targeted by the TaqMan SNP genotyping assay were confirmed by full-genome analysis. Additional events were detected outside the genome regions targeted by the TaqMan assay. The full genomes of the two genotype pattern 9 viruses shared 99.9% pairwise sequence identity. Between these two genotype pattern 9 viruses, 11 SNPs were detected within genes UL[ 1], US5, US6, US7, US8, and US9, located between bp 110000 and bp 140000 in the aligned sequences. Two SNPs were identified in the protein IF and UL49 genes located between bp 8000 to bp 18000 in the aligned sequences, and two SNPs were identified in the UL43 and UL17 genes between bp 74000 and bp 86000. When CSW-1 was used as the reference strain, a total of 55 polymorphisms were found in the six viruses sequenced in this study (see Table S5 in the supplemental material). A high number of recombination breakpoints were identified in two locations extending from bp 20000 to bp 50000 and from bp 106000 to bp 137000 in the six genome sequences from viruses isolated in this study (Fig. 6B). When the full-genome sequences from other ILTV isolates from varied geographical regions were examined, the recombination hot spots detected in those genomes were consistent with those found in the six genomes sequenced from the in vivo study (Fig. 6C, D, and E). Recombination events within genes UL[ 1] and ICP4 were detected in ILTV sequences FIG 2 Legend (Continued) CSW-1 strains of ILTV. (A and B) Low-dose-coinoculated birds F, G, H, I, and J (in the circles) had higher numbers of viral genome copies at day 4 than at day 2 (P 0.05, Mann-Whitney test). These birds did not survive to day 6. (C and D) Low-dose-coinoculated birds K, L, and M (in the rectangles) did not have significantly different numbers of viral genome copies between day 6 and day 8 (P 0.05, Mann-Whitney test). (E and F) High-dosecoinoculated birds A, B, C, D, and E (in the triangles) had higher numbers of viral genome copies at day 4 than at day 2 (P 0.05, Mann-Whitney test). (G and H) High-dose-coinoculated birds did not survive to days 6 and 8. ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 5 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from FIG 3 Recombination patterns, as determined using SNP genotyping, in progeny viruses isolated 2 and 4 days after coinoculation of SPF chickens with 104 PFU of each of CSW-1 and V1-99 ILTV. Each row corresponds to a virus isolate, with the CSW-1 SNPs indicated by gray boxes and the V1-99 SNPs by black boxes. Each distinct recombination pattern was given a unique genotype code (final column). Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 6 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from from Australia and the United States and in our in vivo recombinants (Table 4). Recombination events among the Asian ILTV strains were detected at sites similar to those detected in the Australian strains, with recombination breakpoints found in genes UL4, UL3.5, UL28, UL29, UL30, UL31, UL32, UL33, and UL34 (Table 4). Only one recombination breakpoint was detected and confirmed by several methods in the ILTV sequences from Europe (Italy) (Table 4). Hence, because of the low number of recombination events in the European group, no recombination hot spots were detected. DISCUSSION Our results show that higher levels of diversity in the recombinant progeny occurred at the same time as higher levels of virus replication (as measured by viral titers). A direct relationship between viral replication and recombination diversity would have important implications for disease control, as it suggests that measures taken to reduce viral replication, such as administration of vaccines to limit virus replication following challenge (21), may be able to reduce the diversity of recombinant viruses, thus potentially reducing the capacity of ILTV to evolve to higher levels of virulence. Vaccines are used FIG 4 Recombination patterns, as determined using SNP genotyping, in progeny viruses isolated 2, 4, 6, and 8 days after coinoculation of SPF chickens with 103 PFU of each of CSW-1 and V1-99. Each row corresponds to a virus isolate, with the CSW-1 SNPs indicated by gray boxes and the V1-99 SNPs by black boxes. Each distinct recombination pattern was given a unique genotype code (final column). ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 7 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from regularly in poultry industries to control ILT. As previously described, most vaccines reduce, but do not prevent, ILTV replication (as measured by viral titers) after challenge, due to cell-mediated immune responses that develop in the host in response to vaccination (21). Many vaccine efficacy studies examine ILTV replication after challenge, but some focus only on clinical signs of disease and mortality (21, 22). Furthermore, studies to meet vaccine registration requirements remain focused mainly on mortality and clinical signs of disease (23). Expanding standard vaccine characterization studies to include examination of a vaccine’s capacity to reduce viral replication after subsequent challenge (as measured by viral titers), and potentially viral recombination, would help to further control disease caused by this virus. Similar approaches could be taken for other attenuated live vaccines that are used to control alphaherpesviruses, such as FeHV-1, BoHV-1, and PRV, in veterinary medicine (24, 25) and for varicella-zoster virus (VZV) vaccines in human medicine (26), as these viruses all have capacity for recombination (3). In this study, a high proportion (67%) of viruses detected following in vivo ILTV coinoculation were recombinants. This is consistent with findings from an HSV-1 study that found that 59% of progeny viruses from the cornea and 74% of progeny viruses from the trigeminal ganglia of mice coinoculated with two strains of HSV-1 were identified as recombinants using restriction fragment length polymorphism (RFLP) analysis (13). Additionally, in vitro studies have also shown abundant recombination following alphaherpesvirus coinoculation, although lower proportions of recombinants are typically detected in cell culture than in in vivo models. For example, approximately 26% of progeny viruses obtained after coinoculation of two HSV-1 strains into cultured cells were identified as recombinants using RFLP analysis (13), and 13% of the viral progeny obtained after coinoculation of two VZV strains into cell culture were identified TABLE 1 Genotypes of viruses detected in high-dose-coinoculated (104 PFU) chickens at 2 and 4 days after coinoculationa Day 2 Day 4 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates 4  aFor day 2, there were a total of 42 (46.2%) parental viruses and 49 (53.8%) recombinants, for a total of 91 (100%) viruses; for day 4, there were a total of 16 (20.8%) parental viruses and 61 (79.2%) recombinants, for a total of 77 (100%) viruses. Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 8 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from as recombinants by RFLP analysis (15). Similarly, 10% to 21% of the viral progeny obtained after coinoculation of an FeHV-1 wild-type strain and two vaccine strains were identified as recombinants by restriction endonuclease digestion of PCR products (16). More recently a TaqMan SNP genotyping assay was developed to better detect TABLE 2 Genotypes of viruses detected in low-dose-coinoculated (103 PFU) chickens at 2 and 4 days after coinoculationa Day 2 Day 4 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates 1  there were a total of 38 (53.5%) parental viruses and 33 (46.5%) recombinants, for a total of 71 (100%) viruses; for day 4, there were a total of 29 (35.4%) parental viruses and 53 (64.6%) recombinants, for a total of 82 (100%) viruses. TABLE 3 Genotypes of viruses detected in low-dose-coinoculated (103 PFU) chickens at 6 and 8 days after coinoculationa Day 6 Day 8 Genotype pattern code Chicken(s) from which virus was isolated No. (%) of isolates Genotype pattern code Chicken(s) from which virus was isolated  aFor day 6, there were a total of 15 (34.9%) parental viruses and 28 (65.1%) recombinants, for a total of 43 (100%) viruses; for day 4, there were a total of 31 (57.4%) parental viruses and 23 (42.6%) recombinants, for a total of 54 (100%) viruses. ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 9 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from TABLE 4 Recombination breakpoint analysis of full ILTV genome sequences and field isolates from Asia, Australia, and the United States and in vivo recombinants Origin of isolates Breakpoint (in alignment), 99% CIa Gene(s) at the beginning of the breakpoint locationb Gene(s) at the end of the breakpoint locationb Possible viruses involved in recombination eventc Method(s) by which breakpoint was detected in RDP4 Beginning End Asia (China) 106423–109054 125644–129391  ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 13 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from TABLE 4 (Continued) Origin of isolates Breakpoint (in alignment), 99% CIa Gene(s) at the beginning of the breakpoint locationb Gene(s) at the end of the breakpoint locationb Possible viruses involved in recombination eventc Method(s) by which breakpoint was detected in RDP4 Beginning End In vivo recombinants 53623–68876 120517–121888 UL35, UL36, UL37, UL38, UL39 No gene between UL [ 1] and ICP4 R, sample 23; M, unknown; m, CSW-1 RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 121902–124017 134435–137336 US10, SORF3 US6, US7, US8 R, sample 27; M, V1-99; m, unknown RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 71911–78480 112397–120377 UL42, UL43, UL44, UL21, UL20 ICP4 R, sample 15; M, V1-99; m, CSW-1 RDP, GENECONV, Bootscan, MaxChi, Chimaera, SiScan, 3SEQ 121904–125713 129197–137424 US10, protein SORF3, US2, US3 US4, US5, US6, US7, US8 R, sample 15; M, V1-99; m, sample 23 GENECONV, Bootscan, 3SEQ 74976–97499 111841–114434 UL44, UL21, UL20, UL19, UL18, UL15, UL17, UL14, UL13, UL12, UL11, UL10, UL9 No gene Between UL [-1] and ICP4 R, sample 12; M, sample 1; m, V1-99 RDP, GENECONV, Bootscan, MaxChi, Chimaera, 3SEQ 2256–97499 106970–137424 Entire UL region Entire US region R, sample 12; M, unknown; m, V1-99 GENECONV, MaxChi, Chimaera 1–32193 105020–137424 UL56, protein IF, UL54, UL53, UL52, UL51, UL50, UL49.5, UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22 Entire US region R, sample 12; M, unknown; m, V1-99 GENECONV, MaxChi, Chimaera, 3SEQ 1–1909 5870–17769 No gene UL56, protein IF, UL54, UL53, UL52, UL51, UL50 R, sample 23; M, sample 1; m, sample 12 SiScan 1–53621 107559–120122 UL56, protein IF, UL54, UL53, UL52, UL51, UL50, UL49.5, UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 Entire US region R, V1-99; M, unknown; m, sample 8 GENECONV, MaxChi, SiScan, 3SEQ 1–137424 1–137424 Entire UL region Entire UL region R, sample 12; M, CSW-1; m, sample 27 GENECONV, Bootscan, MaxChi, 3SEQ 1–113123 1–113123 Entire UL region Entire UL region R, sample 27; M, V1-99; m, sample 23 RDP, GENECONV, MaxChi, Chimaera 1–17769 19248–137424 UL56, protein IF, UL54, UL53, UL52, UL51, UL50 UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 R, CSW-1; M, sample 15; m, sample 27 MaxChi, Chimaera, 3SEQ 1–137424 1–137424 Entire UL and US regions Entire UL and US regions R, sample 8; M, sample 15; m, unknown GENECONV, MaxChi, 3SEQ 11775–17921 17923–137424 UL53, UL52, UL51, UL50, UL49.5 UL49, UL48, UL46, UL45, protein IA-IB-IC-ID-IE, UL22, UL24, UL25, UL26, UL26.5, UL27, UL28, UL29, UL30, UL31, UL32, UL33, UL34 R, sample 27; M, sample 8; m, unknown MaxChi, 3SEQ 1–137424 1–137424 Entire UL and US regions Entire UL and US regions R, sample 15; M, sample 1; m, unknown 3SEQ aCI, confidence interval. bUL, unique long; US, unique short. cR, recombinant; M, major parent; m, minor parent. Loncoman et al. Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 14 on November 16, 2017 by guest http://aem.asm.org/
Downloaded from SUPPLEMENTAL MATERIAL Supplemental material for this article may be found at https://doi.org/10.1128/AEM .01532-17. SUPPLEMENTAL FILE 1, PDF file, 2.5 MB. ACKNOWLEDGMENTS We gratefully acknowledge Mahathi Nadimpalli, Nino Ficorilli, and Mesula Korsa for their help with cell culture and Jose Quinteros, June Daly, Jenece Wheeler, and Cheryl Colson for their help with the in vivo studies. This work was supported by the Australian Research Council (FT140101287). Carlos A. Loncoman is supported by Becas Chile, CONICYT, Gobierno de Chile. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. REFERENCES 1. Garcia M, Spatz S, Guy JS. 2013. Laryngotracheitis, p 137–152. In Swayne D, Glisson J, McDougald L, Nolan L, Suarez D, Nair V (ed), Diseases of poultry, 13th ed. Wiley-Blackwell, Hoboken, NJ. 2. Lee SW, Markham PF, Coppo MJ, Legione AR, Markham JF, Noormohammadi AH, Browning GF, Ficorilli N, Hartley CA, Devlin JM. 2012. Attenuated vaccines can recombine to form virulent field viruses. Science 337:188. https://doi.org/10.1126/science.1217134. 3. Loncoman CA, Vaz PK, Coppo MJ, Hartley CA, Morera FJ, Browning GF, Devlin JM. 2017. Natural recombination in alphaherpesviruses: insights into viral evolution through full genome sequencing and sequence analysis. Infect Genet Evol 49:174 –185. https://doi.org/10.1016/j.meegid .2016.12.022. 4. Jetzt AE, Yu H, Klarmann GJ, Ron Y, Preston BD, Dougherty JP. 2000. High rate of recombination throughout the human immunodeficiency virus type 1 genome. J Virol 74:1234 –1240. https://doi.org/10.1128/JVI.74.3 .1234-1240.2000. 5. Holmes EC. 2003. Error thresholds and the constraints to RNA virus evolution. Trends Microbiol 11:543–546. https://doi.org/10.1016/j.tim .2003.10.006. 6. Mahy BWJ. 2010. The evolution and emergence of RNA viruses. Emerg Infect Dis 16:899. https://doi.org/10.3201/eid1605.100164. 7. Crute JJ, Lehman IR. 1989. Herpes simplex-1 DNA polymerase. Identifi- cation of an intrinsic 5=–3= exonuclease with ribonuclease H activity. J Biol Chem 264:19266 –19270. 8. McGeoch DJ, Cook S. 1994. Molecular phylogeny of the alphaherpesvirinae subfamily and a proposed evolutionary timescale. J Mol Biol 238: 9 –22. https://doi.org/10.1006/jmbi.1994.1264. 9. Drake JW, Hwang CBC. 2005. On the mutation rate of herpes simplex virus type 1. Genetics 170:969 –970. https://doi.org/10.1534/genetics.104 .040410. 10. Javier RT, Sedarati F, Stevens JG. 1986. Two avirulent herpes simplex viruses generate lethal recombinants in vivo. Science 234:746 –748. https://doi.org/10.1126/science.3022376. 11. Thiry E, Meurens F, Muylkens B, McVoy M, Gogev S, Thiry J, Vanderplasschen A, Epstein A, Keil G, Schynts F. 2005. Recombination in alphaherpesviruses. Rev Med Virol 15:89 –103. https://doi.org/10.1002/rmv.451. 12. Umene K. 1999. Mechanism and application of genetic recombination in herpesviruses. Rev Med Virol 9:171–182. https://doi.org/10.1002/(SICI) 1099-1654(199907/09)9:3 171::AID-RMV243 3.0.CO;2-A. 13. Kintner RL, Allan RW, Brandt CR. 1995. Recombinants are isolated at high frequency following in vivo mixed ocular infection with two avirulent herpes simplex virus type 1 strains. Arch Virol 140:231–244. https://doi .org/10.1007/BF01309859. 14. Muylkens B, Farnir F, Meurens F, Schynts F, Vanderplasschen A, Georges M, Thiry E. 2009. Coinfection with two closely related alphaherpesviruses results in a highly diversified recombination mosaic displaying negative genetic interference. J Virol 83:3127–3137. https://doi.org/10.1128/JVI .02474-08. 15. Dohner DE, Adams SG, Gelb LD. 1988. Recombination in tissue culture between varicella-zoster virus strains. J Med Virol 24:329 –341. https:// doi.org/10.1002/jmv.1890240310. 16. Fujita K, Maeda K, Yokoyama N, Miyazawa T, Kai C, Mikami T. 1998. In vitro recombination of feline herpesvirus type 1. Arch Virol 143:25–34. https://doi.org/10.1007/s007050050265. 17. Henderson LM, Katz JB, Erickson GA, Mayfield JE. 1990. In vivo and in vitro genetic recombination between conventional and gene-deleted vaccine strains of pseudorabies virus. Am J Vet Res 51:1656 –1662. 18. Loncoman CA, Hartley CA, Coppo MJC, Vaz PK, Diaz-Méndez A, Browning GF, Lee S-w, Devlin JM. 2017. Development and application of a TaqMan single nucleotide polymorphism genotyping assay to study infectious laryngotracheitis virus recombination in the natural host. PLoS One 12:e0174590. https://doi.org/10.1371/journal.pone.0174590. 19. Davison AJ. 2010. Herpesvirus systematics. Vet Microbiol 143:52– 69. https://doi.org/10.1016/j.vetmic.2010.02.014. 20. Vaz PK, Horsington J, Hartley CA, Browning GF, Ficorilli NP, Studdert MJ, Gilkerson JR, Devlin JM. 2016. Evidence of widespread natural recombination among field isolates of equine herpesvirus 4 but not among field isolates of equine herpesvirus 1. J Gen Virol 97:747–755. https://doi.org/ 10.1099/jgv.0.000378. 21. Coppo MJ, Hartley CA, Devlin JM. 2013. Immune responses to infectious laryngotracheitis virus. Dev Comp Immunol 41:454 – 462. https://doi.org/ 10.1016/j.dci.2013.03.022. 22. Coppo MJ, Noormohammadi AH, Browning GF, Devlin JM. 2013. Challenges and recent advancements in infectious laryngotracheitis virus vaccines. Avian Pathol 42:195–205. https://doi.org/10.1080/03079457 .2013.800634. 23. Collet S. 2013. Principles of disease prevention, diagnosis and control introduction, p 4 – 40. In Swayne D, Glisson J, McDougald L, Nolan L, Suarez D, Nair V (ed), Diseases of poultry, 13th ed. Wiley-Blackwell, Hoboken, NJ. 24. Luo Y, Li N, Cong X, Wang C-H, Du M, Li L, Zhao B, Yuan J, Liu D-D, Li S, Li Y, Sun Y, Qiu H-J. 2014. Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China. Vet Microbiol 174:107–115. https://doi.org/10.1016/ j.vetmic.2014.09.003. 25. Jas D, Aeberle C, Lacombe V, Guiot AL, Poulet H. 2009. Onset of immunity in kittens after vaccination with a non-adjuvanted vaccine against feline panleucopenia, feline calicivirus and feline herpesvirus. Vet J 182:86 –93. https://doi.org/10.1016/j.tvjl.2008.05.025. 26. Takahashi M, Otsuka T, Okuno Y, Asano Y, Yazaki T, Isomura S. 1974. Live vaccine used to prevent the spread of varicella in children in hospital. Lancet 304:1288 –1290. https://doi.org/10.1016/S0140-6736(74)90144-5. 27. Kindt R, Coe R. 2005. Tree diversity analysis: a manual and software for common statistical methods for ecological and biodiversity studies. World Agroforestry Centre, Nairobi, Kenya. http://www.worldagroforestry.org/ resources/databases/tree-diversity-analysis. 28. Tóthmérész B. 1995. Comparison of different methods for diversity ordering. J Veg Sci 6:283–290. https://doi.org/10.2307/3236223. 29. Zhao Y, Kong C, Wang Y. 2015. Multiple comparison analysis of two new genomic sequences of ILTV strains from China with other strains from different geographic regions. PLoS One 10:e0132747. https://doi.org/10 .1371/journal.pone.0132747. 30. Agnew-Crumpton R, Vaz PK, Devlin JM, O’Rourke D, Blacker-Smith HP, Konsak-Ilievski B, Hartley CA, Noormohammadi AH. 2016. Spread of the ILTV Recombination in the Natural Host Applied and Environmental Microbiology December 2017 Volume 83 Issue 23 e01532-17 aem.asm.org 17 on November 16, 2017 by guest http://aem.asm.org/ Downloaded from newly emerging infectious laryngotracheitis viruses in Australia. Infect Genet Evol 43:67–73. https://doi.org/10.1016/j.meegid.2016.05.023. 31. Dutch RE, Bruckner RC, Mocarski ES, Lehman IR. 1992. Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences. J Virol 66:277–285. 32. Dutch RE, Bianchi V, Lehman IR. 1995. Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences. J Virol 69:3084 –3089. 33. Lee K, Kolb AW, Sverchkov Y, Cuellar JA, Craven M, Brandt CR. 2015. Recombination analysis of herpes simplex virus 1 reveals a bias toward GC content and the inverted repeat regions. J Virol 89:7214 –7223. https://doi .org/10.1128/JVI.00880-15. 34. Lee S-W, Devlin JM, Markham JF, Noormohammadi AH, Browning GF, Ficorilli NP, Hartley CA, Markham PF. 2013. Phylogenetic and molecular epidemiological studies reveal evidence of multiple past recombination events between infectious laryngotracheitis viruses. PLoS One 8:e55121. https://doi.org/10.1371/journal.pone.0055121. 35. Kirkpatrick NC, Mahmoudian A, O’Rourke D, Noormohammadi AH. 2006. Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes. Avian Dis 50:28 –34. https://doi .org/10.1637/7414-072205R.1. 36. Fahey KJ, Bagust TJ, York JJ. 1983. Laryngotracheitis herpesvirus infection in the chicken: the role of humoral antibody in immunity to a graded challenge infection. Avian Pathol 12:505–514. https://doi.org/10.1080/03079458 308436195. 37. Devlin JM, Browning GF, Hartley CA, Kirkpatrick NC, Mahmoudian A, Noormohammadi AH, Gilkerson JR. 2006. Glycoprotein G is a virulence factor in infectious laryngotracheitis virus. J Gen Virol 87:2839 –2847. https://doi.org/10.1099/vir.0.82194-0. 38. Bagust TJ, Calnek BW, Fahey KJ. 1986. Gallid-1 herpesvirus infection in the chicken. 3. Reinvestigation of the pathogenesis of infectious laryngotracheitis in acute and early post-acute respiratory disease. Avian Dis 30:179 –190. 39. Kirkpatrick NC, Mahmoudian A, Colson CA, Devlin JM, Noormohammadi AH. 2006. Relationship between mortality, clinical signs and tracheal pathology in infectious laryngotracheitis. Avian Pathol 35:449 – 453. https://doi.org/10.1080/03079450601028803. 40. Kawaguchi T, Nomura K, Hirayama Y, Kitagawa T. 1987. Establishment and characterization of a chicken hepatocellular carcinoma cell line, LMH. Cancer Res 47:4460 – 4464. 41. Devlin JM, Browning GF, Gilkerson JR. 2006. A glycoprotein I- and glycoprotein E-deficient mutant of infectious laryngotracheitis virus exhibits impaired cell-to-cell spread in cultured cells. Arch Virol 151:1281–1289. https://doi.org/10.1007/s00705-005-0721-8. 42. National Health and Medical Research Council. 2013. Australian code for the care and use of animals for scientific purposes, 8th ed. National Health and Medical Research Council, Canberra, Australia. 43. Mahmoudian A, Kirkpatrick NC, Coppo M, Lee SW, Devlin JM, Markham PF, Browning GF, Noormohammadi AH. 2011. Development of a SYBR Green quantitative polymerase chain reaction assay for rapid detection and quantification of infectious laryngotracheitis virus. Avian Pathol 40:237–242. https://doi.org/10.1080/03079457.2011.553582. 44. Oksanen J. 2015. Multivariate analysis of ecological communities in R: vegan tutorial. http://vegan.r-forge.r-project.org/. 45. Hill MO. 1973. Diversity and evenness: a unifying notation and its consequences. Ecology 54:427–432. https://doi.org/10.2307/1934352. 46. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/bioinformatics/ bts199. 47. Vaz PK, Job N, Horsington J, Ficorilli N, Studdert MJ, Hartley CA, Gilkerson JR, Browning GF, Devlin JM. 2016. Low genetic diversity among historical and contemporary clinical isolates of felid herpesvirus 1. BMC Genomics 17:704. https://doi.org/10.1186/s12864-016-3050-2. 48. Huson DH, Bryant D. 2006. Application of phylogenetic networks in evolutionary studies. Mol Biol Evol 23:254 –267. https://doi.org/10.1093/ molbev/msj030. 49. Martin DP, Murrell B, Golden M, Khoosal A, Muhire B. 2015. RDP4: detection and analysis of recombination patterns in virus genomes. Virus Evolution 1(1):vev003. https://doi.org/10.1093/ve/vev003.

viernes, 22 de septiembre de 2017

Identification of a divergent genotype of equine arteritis virus from South American donkeys J. Rivas1 | V. Neira2* | J. Mena2 | B. Brito2 | A. Garcia3 | C. Gutierrez3 | D. Sandoval1 | R. Ortega1* 2017

Identification of a divergent genotype of equine arteritis virus from South  American  donkeys


J. Rivas1    |  V. Neira2*       |  J. Mena |  B. Brito2    |  A. Garcia3    |  C. Gutierrez |
D. Sandoval1    |  R. Ortega1*




1Facultad de Ciencias Veterinarias, Departamento de patolog ıa y medicina preventiva,  Universidad  de Concepcion, Chill an, Chile
2Facultad de Ciencias Veterinarias  y Pecuarias,  Universidad  de Chile, Santiago, Chile
3Laboratorio y Estacio  n Cuarentenaria Pecuaria,  Complejo  Lo Aguirre, Servicio Agr ıcola y Ganadero,  Santiago,  Chile

Correspondence
V. Neira, Facultad  de Ciencias Veterinarias  y Pecuarias,  Universidad  de Chile, Santiago, Chile and R. Ortega,  Facultad  de Ciencias Veterinarias,  Departamento de patolog ıa y medicina  preventiva,  Universidad  de Concepcion   , Chill an, Chile.

Present address
B. Brito, Foreign Animal Disease  Research Unit, Plum Island Animal Disease  Center, ARS, USDA, NY, USA


Summary


 
A novel  equine  arteritis  virus (EAV) was  isolated  and  sequenced from feral donkeys in Chile. Phylogenetic analysis  indicates  that  the  new  virus  and  South  African asi- nine  strains  diverged  at  least  100  years  from  equine  EAV strains.  The  results  indi- cate  that  asinine  strains  belonged  to a different EAV genotype.

KEY W ORD S
donkey,  equine,  equine  arteritis  virus, equine  viral arteritis







1    |    INTRODUCTI ON


Equine viral arteritis  (EVA) is a viral disease  in equids,  namely horses, donkeys,  mules  and zebras.  The causative  agent  is the  equine  arteri- tis   virus   (EAV), genus   Equartevirus  from   the   Arteriviridae  family (Adams et  al., 2017).  EAV strains  have  been  classified  based  on  the ORF5 phylogeny  into three  genotypes, the  North  American (NA) and the  European 1 (EU1) and 2 (EU2) lineages  (Zhang et  al., 2007).
Clinical disease  is characterized by  fever  and  respiratory symp- toms;   however,   economic   losses   are  mostly   due   to  its  ability  to cause  abortion  in mares  and  severe  disease  or death  in young  foals (Balasuriya, Go, & MacLachlan, 2013).  EAV increased global reporting during more  recent years has been  attributed to more  frequent international horse  movement (Dominguez,  Munstermann, de  Guin- dos,  & Timoney,  2016).  EVA is not  only transmitted through direct


*These authors should  be considered joint senior  authors.


contact during  clinical respiratory disease,  but  it can  also  be  trans- mitted  through the  venereal  route.  Stallions can become persistently infected  (carriers) and  transmit  the  disease  during  breeding  (Guthrie et  al., 2003).


2    |    MATERIAL S  A ND  METHODS


In Chile, the  EAV has  not  been  detected in horses.  In 2013,  during surveillance  activities,  samples  collected  from  feral  donkeys  ranging in small herds  in hills and  plains  nearby  the  Atacama  Desert were positive   to   neutralizin antibodies  against   EAV  (Moreira,   Garc ıa, Valencia,  & Moreno,  2016).  Following  results  from  this  study,  two male  adult  donkeys,  clinically healthy,  were  captured in the  annual rodeo   event   in  October 2013.   The  rodeo   was  conducted at  Car- rizalillo, Freirina,  Chile  (   29.099469,    71.406169).  Donkeys   were
sent  to a slaughterhouse for human  consumption.



Transbound Emerg Dis. 2017;1–6.                                                   wileyonlinelibrary.com/journal/tbed                                         © 2017  Blackwell Verlag GmbH     1






2.1    Sample  collection

Tissue  samples  including  heart,   lung,  kidney,  testes, vas  deferens, epididymis,  prostate and  seminal  vesicle  were  collected.  One  gram


from each  organ  was  scraped  and  homogenized with  10 ml of mini- mum essential  media (MEM). The mix was centrifuged at 2,823  g for
20 min, and  the  supernatant was  used  for  RT-PCR and  virus  isola-
tion.






 
(a)                                                                     (b)









FI GU RE 1    Cytopathogenic effects  of RK-13 cells: (a) RK-13 cells mock-infected at 7 days post-inoculation. (b) RK-13 cells infected  with Atacama-2014 equine arteritis virus (EAV) isolate  at 7 days post-
inoculation






European 1
European 2
North American
Asinine lineage


U38593.1/Horse/AZ87/Arizona-U.S.A/1987
AF099839.1/Horse/S-436/Poland/1988
AF099850.1/Horse/S1512/U.S.A/1995
EF102379.1/Horse/PLP00-1/Lesser_Poland-Poland/2000
EF102382.1/Horse/PLP02-4/Lesser_Poland-Poland/2002
AY453313.1/Horse/I16/Italy/1995
EF492547.1/Horse/F9/Loire-France/2002
AF099830.1/Horse/3308V-96/Italy/1995
AY359193.1/Horse/H1S/Bekes-Hungary/2001
AY359209.1/Horse/H20F/Heves-Hungary/2000
AY359208.1/Horse/H19F/Pest-Hungary/2000
EF102355.1/Horse/PLH05-1/Silesian-Poland/2005
AY359207.1/Horse/H269S/Zala-Hungary/2003
U38592.1/Horse/AUT68/Vienna-Austria/1968
AF099813.1/Horse/S-1128/Canada/1992
U46952.1/Horse/Vienna/Vienna-Austria/1968
AF099811.1/Horse/Vienna/Austria/1964
EF492558.1/Horse/F20/Ile_de_France-France/2001
AF099814.1/Horse/EAV-86-R/France/1986
GQ903863.1/Horse/S4222/California-U.S.A/2008
AY359197.1/Horse/H147S/Heves-Humgary/2001
AF099815.1/Horse/EAV-86-P/France/1986
AF099823.1/Horse/1192VE4-91/Italy/1990
AF099824.1/Horse/135VE2-95/Italy/1994
AF099838.1/Horse/Wroclaw-2/Poland/1978
AY359201.1/Horse/H197S/Fejer-Hungary/2002


AF099812.1/Horse/Fallat/Canada/1986
U46949.1/Horse/19933/Ontario-Canada/1992
U38594.1/Horse/CAN86/Alberta-Canada/1986
U46948.1/Horse/11958/Ontario-Canada/1990
AF099835.1/Horse/NEAV-1/Norway/1988
AF099836.1/Horse/NEAV-2/Norway/1989
AY359199.1/Horse/H172S/Komarom-Esztergom-Hungary/2002
U38607.1/Horse/PA76/Pennsylvania-U.S.A/1976
EF492549.1/Horse/F11/Ile_de_France-France/2004
EF492543.1/Horse/F5/Basse_Normandie-France/2004
EF492562.1/Horse/F24/Basse_Normandie-France/2004
EF492551.1/Horse/F13/Basse_Normandie-France/2004
JX868590.1/Horse/EAV_HSY/China/2011
U38608.1/Horse/PLD76/Wroclaw-Poland/1976
AF099849.1/Horse/TAQ/U.S.A/1994
U38611.1/Horse/VBS53/Bucyrus-Ohio-U.S.A/1953
AF099842.1/Horse/S-2506/Sweden/1989
U38609.1/Horse/SWZ64/Bibuna-Switzerland/1964
AF099843.1/Horse/Bibuna/Switzerland/1964
AF099828.1/Horse/1330VE-95/Italy/1995
AF099825.1/Horse/470VE1-95/Italy/1994
AF099826.1/Horse/73VE2-95/Italy/1994
AY453340.1/Horse/S2/Sweden/1999
AF099832.1/Horse/1908V-97/Italy/1996
AY349167.1/Horse/CW96/U.S.A/1996
AF118783.1/Horse/CA97/California-U.S.A/1997
AY349168.1/Horse/CW01/U.S.A/2001
AF099816.1/Horse/EAV-86-110/France/1986
AF099822.1/Horse/D526/Germany/1996
AY453344.1/Horse/S6/Sweden/2000
AY453278.1/Horse/A4/Austria/1998
* *                                                                                                                                                                               AY359203.1/Horse/H213S/Hungary/1999
AY359202.1/Horse/H204S/Heves-Hungary/1994
AY359204.1/Horse/H215S/Zala-Hugary/2000
AY359210.1/Horse/H72F/Zala-Hungary/1998
AY359200.1/Horse/H189S/Heves-Hungary/2002
AY453335.1/Horse/RSA4/R.S.A/1996
LC000003.1/Horse/GB_Glos_2012/Gloucestershire-U.K/2012
U38599.1/Horse/KY63/Kentucky-U.S.A/1963
AY956598.1/Donkey/J3-931209/R.S.A/1993
AY956601.1/Donkey/J6-940309/R.S.A/1994
AY956597.1/Donkey/J2-931125/R.S.A/1993
AY956600.1/Donkey/J5-940309/R.S.A/1994
AY956599.1/Donkey/J4-931209/R.S.A/1993
*                                                             Donkey/Atacama-2014/Chile/2014

–700                      –600                      –500                      –400                      –300                      –200                      –100                         0

FI GU RE 2    Maximum clade credibility collapsed  tree  of equine  arteritis  virus using 170  ORF5 reference sequences. The Atacama-2014 and South  African Donkey  sequences belong  to a single monophyletic group, the  asinine  cluster  (red). The time to most  recent common  ancestor (tMRCA) of the  asisine cluster  with other  equine  arteritis  virus (EAV) sequences and the  tMRCA of Atacama-2014 with the  closest  reference are indicated  with * and **, respectively






2.2    RT-PCR

RNA was  extracted using  the  commercial  kit MagMAXTM-96  AI/ND Viral RNA Isolation  Kit (Ambion         Cat#  AM1835,  Austin, TX, USA). ORF6 and ORF7 were  amplified by RT-PCR using the  protocols rec- ommended by the  World  Organisation for  Animal Health  (Timoney,
2012).  ORF5  was  amplified  using  the  primers  and  protocols  previ- ously described (Stadejek et  al., 1999).  PCR products were  submitted for Sanger  sequencing. The positive  samples  were  selected for virus isolation.


2.3    Viral isolation

Viral isolation  was attempted in RT-PCR-positive  samples.  First, monolayers of  RK-13  cells  (ATCC     CCL-37TMwere  grown  in  12- well plates  with cell growth  media, which includes minimum essential medium  Eagles (MEM), supplemented with 10% foetal  bovine  serum,
10,000 IU/ml  penicillin  (1%), 10,000 lg/ml  streptomycin  (1%) and
25 lg/ml  amphotericin B (1%). Monolayers  with  80% of cell conflu- ence  were  inoculated with  200  ll  of  filtrated  positive  samples  and
incubated for  1 hr  at 37°C  and  5% CO2.  After  the  incubation,  the


inoculum  was  discarded and  cells were  incubated for 10 days  using cell  growth   medium   previousl described.   The   monolayers were observed daily during 10 days  inspecting  for evidence  of cytopatho- genic  effects.   Positive  cultures  were  tested by  RT-PCR to  confirm the  presence of the  EAV.


2.4    Phylogeny

ORF5  was  used   to  reconstruct the   EAV phylogeny.   ORF5  is  the most  variable  region  of the  virus and  commonly  used  for EAV phy- logeny.   ORF5  sequence  generated  for   this   study   and   reference sequences covering  the  known  spectrum of  ORF5  genetic  diversity were  aligned  using  MUSCLE (Edgar, 2004).The  codon  partition  and nucleotide  substitution  model   was  selected  using  partition   finder based  on  the  Bayesian  informatio criterion  (BIC) (Lanfear, Calcott, Ho,  & Guindon,  2012).  The  best  scheme  consisted in one  partition for  each  codon  position:  HKY+I+G  for  codon  position  1,  TrN+I+G for codon  position  2 and  GTR+I+G for codon  position  3. We  used  a Bayesian  approach for  time  divergence estimation  implemented  in BEAST 1.8.2  (Drummond  & Rambaut,  2007).  Initially, a  strict  clock
model  and  an  uncorrelated relaxed  lognormal  clock, in combination







GQ903859/2007/USA/S3901
GQ903809/2005/USA/S3583
GQ903901/2005/USA/M405
GQ903811/2008/USA/S4216
EF492559/2002/France/F21
AF118780/1998/USA/G4
AF118777/1995/USA/G1
AF118779/1997/USA/G3
AF118778/1997/USA/G2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118776/1997/USA/P2
AF118775/1996/USA/P1
AF118774/1998/USA/R2
AF118773/1996/USA/R1
AF118769/1995/USA/A1
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
EF492556/2001/France/F18
EF492557/2001/France/F19
EF492555/2001/France/F17
EF492560/2002/France/F22
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492548/2003/France/F10
EF492553/2001/France/F15
JN211317/2000/France/F60
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01


European 1
European 2
North American
Atacama-2014


HK25
Hela-EAVP35
EF492564/2004/France/F26
EF492563/2004/France/F25
EF492562/2004/France/F24
U81013/1953/USA/VBS53
U81020/NA/USA/ATCC
EF492552/2003/France/F14
EF492550/2004/France/F12
EF492549/2004/France/F11
EF492545/2004/France/F7
EF492544/2004/France/F6
U81019/NA/USA/ARVAC
pEAVrMLV
EF492543/2004/France/F5
EF492546/2004/France/F8
EF492551/2004/France/F13
DQ846750/1953/USA/Bucyrus
U81021/1986/Canada/CAN86
U81018/1976/USA/PA76
AH007128/1999/USA/CA95G
GQ903862/2007/USA/S3955
AF107267/1999/USA/D85
AF107271/1999/USA/D89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107272/1999/USA/D91
AF107276/1999/USA/E86
AF107268/1999/USA/D86
AF107277/1999/USA/E88
AF107278/1999/USA/E89
AH007129/1999/USA/CA95I
U81015/1977/USA/KY77
U81017/1993/USA/KY93
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107266/1999/USA/D84



















Donkey/Atacama-2014/Chile/2014



0.08

FI GU RE 3    Maximum likelihood ORF6 phylogenetic tree  using 93 equine  arteritis  virus reference sequences. Atacama-2014 isolate  is a singleton  genetically  distant  from reference sequences coloured  in red



with  a constant  population and  a Bayesian  skyline  tree  prior,  were run.   We   selected  the   model   based   upon   the   AICm  method-of- moments estimator (Baele et  al., 2012;  Raftery,  Newton, Satagopan,
& Krivitsky, 2007)  implemented  in  Tracer  v1.6  (Rambaut,  Suchard, Xie, & Drummond,  2014).  Based  on  the  lower  AICm, the  uncorre- lated  exponential clock model  and  a coalescent constant population tree  prior were  selected. The analysis was run for 200,000 iterations. Convergence and  mixing of the  simulations  was  assessed using Tra- cer. The maximum clade credibility tree  (MCC) was visualized in Fig- tree    version    1.4.2    (Rambaut,    2014).    Additionally,   phylogenetic analyses  of ORF6 and ORF7, which are more  conserved genes,  were performed  using  MUSCLE for  sequence  alignment   and  maximum likelihood to  reconstruct the  phylogeny  in MEGA7 (Kumar, Stecher,
& Tamura, 2016).


3    |    RESULTS  A ND  DI SCUSSI ON


Viral RNA was  identified  in samples  from  both  animals. From  a vas deferens  sample,   cytopathogenic  effects   (CPE) were   observed at
5 days  post-inoculation. CPE was  characterized by rounding  of cells and  cell  detachment  from  the   monolayer   (Figure 1).  The  isolated virus was named  Atacama-2014.
Viral RNA was  amplified,  and  partial  sequences of  ORF5  (630 nucleotides- nt), ORF6  (193  nt)  and  ORF7  (316  nt)  were  obtained by Sanger sequencing method (Accession numbers MF543058, MF543059 and  MF573786,  respectively).  The  phylogeny  of  ORF5 revealed   that  the  Atacama-2014 belonged   to  a  monophyletic  clus- ter   that   included   viruses   collected   from   donkeys   in  1993–1994
sample (Stadejek,   Mittelholzer,    Oleksiewicz,   Paweska,   &  Belak,








GQ903809/2005/USA/S3583
GQ903811/2008/USA/S4216
GQ903901/2005/USA/M405
GQ903859/2007/USA/S3901
EF492556/2001/France/F18
AF118779/1997/USA/G3
AF118780/1998/USA/G4
AF118778/1997/USA/G2
AF118777/1995/USA/G1
EF492557/2001/France/F19
EF492559/2002/France/F21
EF492555/2001/France/F17
AF118769/1995/USA/A1
AF118773/1996/USA/R1
AF118774/1998/USA/R2
AF118782/1996/USA/RQ
AF118781/1996/USA/BT-PA96
AF118770/1996/USA/A2
AF118771/1996/USA/A3
AF118772/1997/USA/A4
AF118775/1996/USA/P1
AF118776/1997/USA/P2
EF492554/2001/France/F16
EF492561/2002/France/F23
EF492560/2002/France/F22
EF492553/2001/France/F15
JN211317/2000/France/F60
EF492548/2003/France/F10
JN211316/2007/France/F27
AY349167/1996/USA/CW96
AY349168/2001/USA/CW01
GQ903862/2007/USA/S3955
AF107266/1999/USA/D84
AF107267/1999/USA/D85
AF107277/1999/USA/E88
AF107276/1999/USA/E86
AF107275/1999/USA/E85
AF107279/1984/USA/KY84
AF107278/1999/USA/E89
AF107269/1999/USA/D87
AF107270/1999/USA/D88
AF107268/1999/USA/D86
AF107272/1999/USA/D91
AF107273/1999/USA/D92
AF107274/1999/USA/D94
AF107271/1999/USA/D89
U81015/1977/USA/KY77
AH007128/1999/USA/CA95G
AH007129/1999/USA/CA95I
U81018/1976/USA/PA76
U81017/1993/USA/KY93
DQ846750/1953/USA/Bucyrus
U81020/NA/USA/ATCC
EU252113/NA/USA/Hela-EAVP35
U81013/1953/USA/VBS53
EF492564/2004/France/F26


European 1
European 2
North American
Atacama-2014































Donkey/Atacama-2014/Chile/2014




0.03

FI GU RE 4    Maximum likelihood ORF7 phylogenetic tree  using 93 equine  arteritis  virus reference sequences. Atacama-2014 isolate  is a singleton  genetically  distant  from reference sequences coloured  in red





2006);  we  named  this  group  the  asinine  cluster.  The  time  to  most recent common  ancestor (tMRCA) between the  asinine  cluster  and other   EAV genotypes was  estimated at  1695   (95%  highest  poste- rior  density   (HPD)  interval   1424–1892)  (Figure 2,  Appendix   S1). Additionally,  the   tMRCA  of  the   Atacama-2014 sequence  and  the African  donkey   strains   was  estimated  at  1914   (95%  HPD  1779
1983).  The  ORF5  sequence  with  the  highest   identity   to  the  Ata- cama-2014 virus  (78.9%) that  was  public  available  was  J2-931125
#AY9565   (South   African  asinine   cluster).   The  ORF5  genetic   dis- tance   between  groups   (NA,  EU1,  EU2  and   asinine)   was   higher between  the   asinine   group   and   all  other   clusters   (79.5%–81.9%) compared  to  the   remaining  distances between  the   European and North   American   clusters   (90.1%–89.1%).   In  South   America,  only EAV sequences from  Argentina  are  available  from  public  reposito- ries.  These  Argentinean sequences  were  collected  during  the  early
2000s and  in 2011  and  have  been  classified  within  the  EU1 geno- type,  genetically  distant  to  the  asinine  cluster  (Metz,  Serena,  Panei, Nosetto, & Echeverria,  2014).
No  ORF6  and  ORF7  sequences  from  the   donkey   South   Afri- can   isolates   were   available  for  phylogenetic  analysis.  The  ORF6 and  ORF7  phylogeny  shows  the  Atacama-2014 virus  phylogeny  as a  singleton distant   from  all  other   EAVs (Figures 3  and  4).  How- ever,   we   were   able   to   sequence  only  three   ORFs  of  the   virus (10%  genome),  which   we   consider   the   major   limitation   of   this study.
The  phylogeny   indicates   that   the   asinine   cluster   represents  a new  genotype present in South  America and  Africa, both  related  to carrier  donkeys.  The presence of this genotype in two  different con- tinents may  underlie   a  widely  distributed unreported  existence of this  viral  strain,  different  from  the   known   and  well-characterized EAV prevalent in horses.
It is not  clear  how  the  virus was  introduced into  the  feral  don- key  population in Chile. Historical  records  indicate  that  the  original population of  donkeys   arrived  into  the  country   at  least  500  years ago.  However,   it  is  likely  that   subsequent  importations occurred. tMRCA  of  the  Chilean  and  1993   South  African  donkey   was  esti- mated  between 1779  and  1983,  suggesting  that  the  introduction of the  virus may have  occurred during  imports  after  the  original intro- duction  of donkeys  in Chile. However,  because of the  lack of avail- ability  of  viral  sequences  collected   at  earlier  time   points   of  the asinine   cluster,   the   tMRCA  estimates  should   be   carefully   inter- preted.
Although  EAV has  only  been  detected in donkeys  in Chile, the South  African asinine  strains  have  also detected in horses  (Stadejek et  al., 2006);  therefore, the  EAV Chilean  donkey  viruses  may repre- sent  a risk for different equine  populations.
The  characterization  of  this  virus  in  South  America  provides  a novel  perspective of  the  global  distribution  of  EAV. The  isolation and genetic  characterization of this new  virus provides  vital informa- tion for future  EAV surveillance.  It further  contributes to understand the  divergence of  the  virus  and  to  the  proper  design  of  diagnostic test   for  more   accurate detection  in  horses   and  as  well  as  other equids.


AC KN O W L E D G EM E N T S

We  thank  the  staff  of  Chilean  Agricultural  and  Livestock  Services (SAG) for all their support during the  sampling, necropsy  and virology work.


CONFLI CT  OF  INT E RE ST

The authors declare  no conflict of interest.


OR CI D




REF E RE NCE S

Adams, M. J., Lefkowitz, E. J., King, A. M. Q., Harrach,  B., Harrison,  R. L., Knowles,  N. J., ... Davison,  A. J. (2017).  Changes  to  taxonomy  and the  international code  of  virus  classification  and  nomenclature  rati- fied by the  International Committee on taxonomy  of viruses.  Archives of  Virology, 162,  2505–2538.  https://doi.org/10.1007/s00705-017-
Baele,  G., Lemey, P., Bedford,  T., Rambaut,  A., Suchard,  M. A., & Alek- seyenko,  A. V. (2012).  Improving  the  accuracy  of  demographic and molecular   clock  model   comparison  while  accommodating  phyloge- netic   uncertainty.  Molecular  Biology and  Evolution, 29,  2157–2167. https://doi.org/10.1093/molbev/mss084
Balasuriya, U. B. R., Go, Y. Y., & MacLachlan, N. J. (2013). Equine arteritis virus. Veterinary Microbiology, 167, 93–122.  https://doi.org/10.1016/j. vetmic.2013.06.015
Dominguez,  M., Munstermann, S., de  Guindos,  I., & Timoney,  P. (2016).
Equine  disease  events resulting  from international horse  movements: Systematic review  and  lessons  learned.  Equine Veterinary Journal,  48,
Drummond,   A. J., & Rambaut,  A. (2007).  BEAST: Bayesian  evolutionary analysis  by  sampling  trees.   BMC Evolutionary Biology, 7,  214. https://doi.org/10.1186/1471-2148-7-214
Edgar,  R. C.  (2004).  MUSCLE: Multiple  sequence  alignment   with  high accuracy   and   high  throughput.  Nucleic  Acids Research,  32,  1792
Guthrie,  A. J., Howell,  P. G., Hedges,  J. F., Bosman,  A. M., Balasuriya, U.
B. R., McCollum,  W.  H., ... MacLachlan,  N. J. (2003).  Lateral  trans- mission  of  equine  arteritis  virus  among  Lipizzaner  stallions  in South Africa.  Equine  Veterinary  Journal,  35,  596–600.  https://doi.org/10.
Kumar, S., Stecher,  G., & Tamura,  K. (2016).  MEGA7: Molecular  Evolu- tionary  Genetics   Analysis version  7.0  for  bigger  datasets. Molecular Biology and Evolution, 33(7), 1870–1874. msw054. https://doi.org/10.
Lanfear,  R., Calcott,  B., Ho, S. Y. W., & Guindon,  S. (2012).  PartitionFin- der:  Combined   selection   of  partitioning   schemes  and  substitution models  for phylogenetic analyses.  Molecular Biology and Evolution, 29,
Metz,  G. E., Serena,  M.  S., Panei,  C. J., Nosetto,  E. O.,  & Echeverria, M.  G.  (2014).   Th equin arteriti virus  isolate   from   the  2010
Argentinia outbreak.  Revue  Scientifiqu et  Technique,  33 937
946.
Moreira,  R., Garc ıa, A., Valencia,  J., & Moreno,   V. (2016).  Equine  Viral Arteritis in feral donkeys  (Equus asinus) of the  Atacama  Region, Chile. Journal of Equine Veterinary Science, 39, S63–S64.  https://doi.org/10.





Raftery, A. E., Newton, M. A., Satagopan, J. M., & Krivitsky, P. N. (2007).
Estimating  the  integrated likelihood via posterior simulation  using the harmonic  mean  identity.  In J. M. Bernardo,  M. J. Bayarri & J.O. Ber- ger  (Eds.), Bayesian  Statistics  (pp.  1–45).  Oxford,   United   Kingdom: Oxford  University  Press.
Rambaut,  A. (2014). FigTree version  1.4.2 [computer program].
Rambaut,  A., Suchard, M., Xie, D., & Drummond,  A. (2014). Tracer version
1.6 [computer program].
Stadejek,  T., Bjorklund, H., Bascun~ana,  C. R., Ciabatti,  I. M., Scicluna, M.
T., Amaddeo,   D.,  .. .  Bel ak,  S.  (1999).  Genetic   diversity  of  equine arteritis   virus.  Journal  of  General  Virology, 80(Pt  3),  691–699. https://doi.org/10.1099/0022-1317-80-3-691
Stadejek,  T., Mittelholzer,  C., Oleksiewicz,  M. B., Paweska,  J., & Bel ak, S. (2006).  Highly diverse  type  of  equine  arteritis  virus  (EAV)  from  the semen  of a South  African donkey:  Short  communication. Acta Veteri- naria   Hungarica 54,   263–270.  https://doi.org/10.1556/AVet.   54.
Timoney, P. J. (2012). Chapter  2.5.10.  Equine viral arteritis.  In OIE manual of  diagnostic  tests  and  vaccines  for  terrestrial  animals  (7th  edn,  pp.
899–912).  Paris, France:  Office International des Epizooties.


Zhang, J., Miszczak, F., Pronost,  S., Fortier, C., Balasuriya, U. B. R., Zientara, S., ... Timoney, P. J. (2007). Genetic variation and phylogenetic analysis of 22 French isolates  of equine  arteritis  virus. Archives of Virology, 152,



SUPPO R TING  INFORMATION

Additional  Supporting  Information  may  be  found  online  in the  sup- porting  information  tab for this article.




How to cite this article:  Rivas J, Neira V, Mena  J, et  al. Identification of a divergent genotype of equine  arteritis  virus from South  American donkeys.  Transbound Emerg Dis.