Journal MVZ Cordoba 2020; 25(3):e1875. https://doi.org/10.21897/rmvz.1875 Original Replication and survival traits of spring viremia of carp virus (SVCV) isolated in Mexico Leticia Cañas L1 MVZ; Sandra Hernández-Dávila1 Biól; Juan Carlos Vázquez-Chagoyan1 Ph.D; Simón Martínez-Castañeda1 Ph.D; Raúl Fajardo M1 Ph.D; Benjamín Valladares-Carranza1 Ph.D; César Ortega S1* Ph.D. 1Universidad Autónoma del Estado de México, Facultad de Medicina Veterinaria y Zootecnia, Centro de Investigación y Estudios Avanzados en Salud Animal (CIESA). Carretera Toluca-Atlacomulco Km. 15.5, CP 50200. Toluca, México. Correspondencia: [email protected] Received: December 2019; Accepted: May 2020; Published: August 2020. ABSTRACT Objective. To perform the isolation of spring viremia of carp virus (SVCV) in common carp (Cyprinus carpio) and evaluate its growth in different cell types and viral survival at different temperatures. Materials and methods. Ten carps of between 400-500 grams of a lagoon in central Mexico were processed for diagnosis of SVCV by isolation in cell culture and by RT-PCR. The virus obtained was inoculated into EPC, BF-2, CHSE-214 and RTG-2 cells to determine differences in virus growth; the survival of virus stored at room temperature (TA 20-25°C), refrigeration (REF 4°C) and freezing (CONG -80°C) up to eleven months was also evaluated. Internal organ samples were processed for histological analysis. Results. The fish analyzed did not show external signs suggestive of disease but internally and histopathologically lesions suggestive of systemic infection were observed. SVCV was isolated in EPC and BF-2 cells and confirmed by semi-nested RT-PCR. SVCV only induced CPE in EPC and BF-2 cells and was negative in RTG-2 and CHSE-214. The virus conserved at TA lost viability after four months post-infection (mpi), being total at six mpi; while REF and CONG were stable during the eleven months. Conclusions. Subclinical SVCV infection was confirmed in carp that presented histological lesions associated with this infection; SVCV only caused CPE in EPC and BF-2 cells; and the virus kept in refrigeration and at -80°C retained its viability up to eleven months; while TA was lost in six months. Keywords: Cyprinus carpio; fish; disease; RT-PCR; infection; cells (Source: AGROVOC). RESUMEN Objetivo. Realizar el aislamiento del virus de la viremia primaveral de la carpa (SVCV) en ejemplares de carpa común (Cyprinus carpio), evaluar su crecimiento en diferentes tipos de células, así como la supervivencia viral a diferentes temperaturas. Materiales y métodos. Diez carpas de entre 400- 500 gramos de una laguna del centro de México fueron procesadas para el diagnóstico de SVCV mediante aislamiento en cultivo de células y RT-PCR semianidado. El virus obtenido se inoculó en células EPC, BF-2, CHSE-214 y RTG-2 para determinar diferencias de crecimiento de SVCV. Además, se evaluó la supervivencia del virus conservado a temperatura ambiente (TA 20-25°C), refrigeración How to cite (Vancouver). Cañas LL, Hernández-Dávila S, Vázquez-Chagoyan JC, Martínez-Castañeda S, Fajardo MR, Valladares-Carranza B, Ortega SC. Replication and survival traits of spring viremia of carp virus (SVCV) isolated in Mexico. Rev MVZ Cordoba. 2020; 25(3):e1875. https://doi.org/10.21897/rmvz.1875 ©The Author(s), Journal MVZ Cordoba 2020. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by-nc-sa/4.0/), lets others remix, tweak, and build upon your work non-commercially, as long as they credit you and license their new creations under the identical terms. ISSNe: 1909-0544 Cañas et al - Replication traits of SVCV isolated in Mexico (REF 4°C) y congelación (CONG -80°C) hasta once meses. Los órganos internos se procesaron para análisis histológico. Resultados. Los peces analizados no presentaron signos externos sugestivos de enfermedad, pero interna e histopatológicamente se observaron lesiones sugestivas de infección sistémica. SVCV fue aislado en células EPC y BF-2 y confirmado por RT-PCR semianidado. SVCV únicamente indujo CPE en células EPC y BF-2 y fue negativo en RTG-2 y CHSE-214. El virus conservado a TA perdió viabilidad después de cuatro meses post infección (mpi), siendo total a seis mpi; mientras REF y CONG fueron estables durante los once meses de estudio. Conclusiones. La infección subclínica por SVCV fue confirmada en carpas que presentaron lesiones histológicas asociadas a esta infección. SVCV únicamente causó CPE en células EPC y BF-2 y el virus conservó su viabilidad a 4ºC y -80°C hasta once meses; mientras que a TA se perdió en seis meses. Palabras clave: Cyprinus carpio; peces; enfermedad; RT-PCR; infección; células (Fuente: AGROVOC). INTRODUCTION across Europe (1,2), the Middle East (4), and China (2). In America, SVC has been reported in Spring viremia of carp (SVC) is a systemic the USA (2,3), as well as Canada and Brazil (2). infectious disease that primarily affects cyprinids This disease has not previously been officially and is listed as a required notifiable disease by recognized in Mexico (5,6). the Office International des Epizooties (OIE) of the World Organization for Animal Health. SVC virus (SVCV) has been replicated in primary This disease is considered one of the primary cultures of the gonads and swim bladder of economic and health risks for carp (Cyprinus carp, in various fish cell lines, and, even, in spp.) maintained under intensive farming cell lines of other vertebrates (1,2). However, conditions. In wild fish, SVC generally presents epithelioma papulosum cyprini (EPC) and sub-clinically, meaning that infection can go fathead minnow (FHM) cells are the primary unnoticed (1,2). candidates (1,2). Furthermore, SVCV presents differences in growth and survival under different SVC principally occurs in young fish (i.e. <1 year) temperature conditions. According to the OIE and can reach mortality rates of 90%. Mortality Manual of Diagnostic Tests for Aquatic Animals and clinical signs can be influenced by a number (2), SVC should be diagnosed through more of factors, notable among which are the age of than one technique. In clinically affected fish, the fish, the presence of secondary infections, and these techniques include viral isolation or direct the water temperature (2). The most common detection in tissues by the indirect fluorescent clinical signs occur at temperatures between 10 antibody technique (IFAT), the enzyme-linked and 17°C. Accumulated mortality can likewise immunosorbent assay, or polymerase chain occur in this temperature range, with the fastest reaction (PCR). Furthermore, direct identification mortalities occurring at 17°C (1,2). should be confirmed through isolation plus neutralization or through RT-PCR together with Septicemic infection in fish evidences sequencing of the obtained product. nonspecific clinical symptoms, such as pale gills; exophthalmia; hemorrhaging and petechiae Regarding fishing output in Mexico, carps of the skin, gills, and eyes; and abdominal (Cyprinus spp.) rank 8th in terms of volume (i.e. distention. Necropsy can further reveal serous 53,421 tons) and 14th in terms of economic value fluid or fluid mixed with blood or necrotic (7). Since being introduced to Mexico, carps have material; focal hemorrhages or petechiae in the been distributed in bodies of water across nearly muscle and fat tissue, swim bladder, and other the entire country, with the primary purpose abdominal organs; and a usually swollen, thickly being extensive production (7,8,9). Regarding textured spleen (1). Chronic cases of infection sanitary concerns, reports of clinical disease present pleural adherences between internal are rare, and no studies exist determining organs. Infected animals that do not die or those the health status in Mexican carps based on a that are infected without clinical disease act as comprehensive health diagnosis (5). carriers (2,3). While specimens did not present signs of disease, SVC was first reported in Yugoslavia in 1970 (1) SVCV was initially reported by our group in and has since been reported in various countries common carp (Cyprinus carpio carpio) from a Rev MVZ Córdoba. 2020 September December; 25(3):e1875 2/8 https://doi.org/10.21897/rmvz.1875 Cañas et al - Replication traits of SVCV isolated in Mexico lagoon in central Mexico at the end of 2015 (5). at 20°C for 7 days to determine which cells Despite the results of isolation (5), the respective evidenced viral multiplication. health authorities ruled out the presence of a virus through molecular analysis based only on Survival of SVCV under maintenance at RT-PCR. As such, SVCV is still considered exotic three temperatures. The supernatant from for Mexico (6). For the present study, new carp primary isolate cultures was divided into three specimens were collected, and cell culturing aliquots. One was kept at room temperature and semi-nested PCR were used to detect the (i.e. 20-25°C), another under refrigeration (i.e. presence of SVCV in Mexican carp. Additionally, 4°C), and another under freezing conditions (i.e. described are the growth traits of the virus in -80°C). Survival of the conserved virus over different cell lines and viral survival at different an 11-month period (i.e. March 206 to January temperatures. 2017) at different temperatures was assessed by determining the maximum dilution at which the virus was able to induce the CPE. The viral MATERIALS AND METHODS titer was calculated using serial dilutions (10-1 to 10-8) in EPC cells at 90% confluence on a 96- Sample collection. In October 2015, the well plate. The plate was sealed and incubated aquatic animal health authorities used molecular at 25°C, and the presence or absence of the CPE diagnosis to rule out the presence of SVCV in was recorded up to 7 days post-inoculation to the Tecocomulco lagoon where the virus has determine viral titer.