Effect of temperature on the expression of IFN-1 (α), STAT-1 and Mx-1 genes in Oncorhynchus mykiss (Salmoniformes: Salmonidae) exposed with the virus of the infectious pancreatic necrosis (IPNV)
Efecto de la temperatura sobre la expresión de genes IFN-1 (α), STAT-1 and Mx-1 en alevines de trucha arcoíris Oncorhynchus mykiss (Salmoniformes: Salmonidae) expuestos al el virus de la necrosis pancreática infecciosa (IPNV)
Donald Arguedas Cortés1*,4*, Alex P. Romero Zuñiga2*,3*, Ricardo Enriquez Sais2, José S. Martínez Castañeda1 & César Ortega Santana1
Abstract
The infectious pancreatic necrosis (IPNV) is the causative agent of an acute illness well characterized in salmonids worldwide. Clinical signs and mortality rates are dependent on several factors such as the viral dose, the age of the fish, the water temperature, among others. An experimental study was conducted to measure the effect of temperature on the gene expression profile of IFN-1(α), STAT-1 and Mx-1 in rainbow trout fry, exposed to IPNV. Fry ]]>
The virus of infectious pancreatic necrosis (IPNV) is an aquabirnavirus member of the Birnaviridae family that causes infectious pancreatic necrosis (IPN) (OIE, 2006), an emerging disease that affects predominantly salmonids (Roberts, & ]]>
Salvelinus fontinalis) in North America (M’gonigle, 1941), however, the agent was not isolated from clinically ill animals until 1957 (Wolf, Snieszko, Dunbar, & Pyle, 1960).
IPNV has a cosmopolitan distribution that causes variable mortality rates worldwide (Ortega et ]]>
The clinical illness episode and the mortality percentage have been reported to depend on various ]]>
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The IPNV genome is composed of two segments of double-stranded RNA (dsRNA) that encodes five viral proteins. The segment A encodes VP2, VP3, VP4 and VP5; while B encodes only VP1. These proteins can activate or inhibit certain defense mechanisms of the immune system including the expression of Interferon-stimulated genes (ISGs) type1, considered the first line of defense against viral infection (Robertsen, Bergan, Rokenes, Larsen, & Albuquerque, 2003; Zhou et al., 2007). ]]>
The IFN-1(α) activation triggers the signaling pathway JAK-STAT by inducing genes such as Mx, 2-5 oligo adenylate synthetase (2-5 OAS), viperine antiviral protein (Vig-1), kinase protein RNA (PKR), among others (Sen, 2001; Platanias, 2005; Skjesol, Toril, Hegseth, Børre, & Jørgensen, 2009). JAK kinases pathway JAK-STAT (JAK1 and TYK2) and transcription factors associated with the family of STAT proteins (STAT-1, 2, 3 and 4) (Zhou et al., 2007) have been well studied in mammals (Sen, 2001) but poorly ]]>
Innate immunity is developed from embryogenesis as a faster response, unlike the adaptive response that is performed four to six weeks after fertilization (Herbomel, Thisse, & Thisse, 2001). It is independent of temperature and the most important defense mechanism in aquatic organisms (Robertsen, 2006). Rubio-Godoy (2010) prefers to refer to it as relatively independent of ]]>
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It is well known that change in water temperature has an impact on rainbow trout physiology (Guderley, 2004; Kraffe, Marty, & Guderley, 2007; Haverinen, & Vornanen, 2007) including their metabolic rate (Battersby, & Moyes, 1998). Additionally, immune-related functions such as endocytosis (Padron, Bizeau, & Hazel, 2000), immune gene transcription (Raida, & Buchmann, 2007), immunoglobulins concentration (Suzuki, Otaka, Sato, Hou, & Aida, 1997; Nikoskelainen, Bylund, & Lilius, 2004), and C-reactive protein (Kodama et al., 2004) have been reported to be ]]>
The present work contributes to our understanding of temperature effects on the expression of relevant antiviral genes of innate response in rainbow trout following bath exposed with IPNV.
Materials and Methods
Fish: A total of 198 rainbow trout fry (Oncorhynchus mykiss) (average weight of 1.0g±0.18 and a length of 3.92cm±0.14) were obtained from a farm with an IPNV- free history and transported to the Virology Laboratory, Research Center in Animal Health (CIESA),Toluca, Mexico. Before the experiment ]]>
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Virus: An isolated IPNV (strain Buhl), similar to the one obtained by Ortega, et al. (2002) and supplied by CIESA, was replicated by inoculation in bottles of 500mL in Chinook salmon embryo cells (CHSE-214) with 90% confluence maintained in (MEM), supplemented with 100µg/mL of streptomycin, 60µg/of penicillin and 2% of (FBS). Once reaching an extensive cytopathic effect (EPC), the virus was titered using the microplate method (Reed & Muench, 1938). ]]>
Infection: Fish (average weight of 1.0g±0.18 and a length of 3.92cm±0.14) were exposed during 30 minutes with a viral solution of 1x106.16 TCDI50/mL of MEM (endpoint dilution assay quantifies the amount of virus required to kill 50% of infected to produce a cytopathic effect in 50% of inoculated tissue culture cells). Afterwards, they were transferred into three other glass tanks (with water temperatures at 8, 12 and ]]>
ad libitum (12% of body mass/day) with a commercial feed for trout (El Pedregal TM). The water used in each of the glass tanks was chlorinated after the experiment to destroy viruses (OIE, 2006).
Samples: At days 2, 4, 7, 11, 14 ]]>
Extracting total RNA, Retrotranscription (RT): Total RNA was extracted from kidney using the commercial kit (E.Z.N.ATM Total RNA Kit I, Omega-Biotek) and according to the manufacturer’s instructions. A sample of 9.4 µL of RNA was incubated using a thermocycler (Labnet Multigene Gradient) at 37°C for 30minutes, and was treated with DNase (Promega Cat.#M6101), mixed with 1µL of RQ1 RNase-Free DNase and 1µL RQ1 DNase 10X Reaction Buffer per sample. Immediately, 1 µL of Stop Solution was added, and the samples were incubated at 65°C for 10 minutes. ]]>
Real-Time PCR (qRT-PCR): The cDNA was amplified with a Step OneTM Real Time PCR system Thermal Cycling Block (Applied Biosystems) using the SYBR®Green method. PCR amplification was performed in individual wells of a 48-well optical plate, mixing 2µL of cDNA, 1µL primer Forward, 1µL ]]>
We performed Pearson’s correlation (r) test to determine the strength of association between viral titer (n=5) and time (dpi), and viral titer with gene expressions (fold). ]]>
Effect of the temperature on average gene expression in exposed fish along the study: On average, expression profile of IFN-1(α) was significantly highest in fish exposed at 16ºC (Tukey, p<0.0005), while the temperature did not exert significant effect on STAT-1 expression average (Tukey, p>0.05); although, the highest average level expression was recorded at 16°C. At 12ºC, Mx-1 showed a highly significant increase in average level of expression (Tukey, p<0.0001).
Gene expression at 8°C: The IFN-1(α) gene expression was lower when compared with values obtained at 16ºC (Fig. 2A), but was higher at days 7 and 21, when compared with those obtained at 12 and 16ºC. STAT-1 showed a significant up-regulation on day 4 when compared with the values registered at 12 and 16ºC, and on day 7 compared with those registered at 12°C (Fig. 2B) (t-student, p<0.05). Besides, on day 14, a down-regulation was registered compared with the values obtained at 12°C. It was evident that with the increase of viral titer, the expression of this gene also increased (r=0.91). The Mx-1 expression profile showed a significant up-regulation (~ 21.8 fold) at days 4, 11 and 14 when compared with IFN-1 (α) and STAT-1 values (Fig. 3A) (t-student, p<0.05). A highly positive association between viral titer values and Mx-1 expression profile (r=0.96) was observed. The animals showed some symptoms of IPN disease, however recovered from the symptoms presented from day 4. ]]>
Gene expressions at 12°C: The detected IFN-1(α) showed lower expression when compared with the values detected at 16ºC, but this transcript was practically inhibited at day 7 (Fig. 2A) (t-student, p<0.001). The STAT-1 expression value on day 7 was significantly inhibited at 12°C as compared to the values ]]>
Fig. 2B) (t-student, p<0.05). Additionally, we observed that the expression profile of STAT-1 was lower on day 2 and 4 when compared with Mx-1 value, and completely down-regulated on day 7 (~ 0.020 fold). Furthermore, we observed that STAT-1 expression profile was lower on days 4 and 14 compared to the value registered for IFN-1(α) (Fig. 3B) (t-student, p<0.0001). It ]]>
Fig. 3C) (t-student, p<0.05). The Mx-1 expression profile was highest at days 2, 4 and 7 when compared with IFN-1(α) and STAT-1, showing a highly substantial up-regulation of transcript on day 4 (Fig. ]]>
) (t-student, p<0.0001) and day 14 (t-student, p<0.05). We found a negative correlation between viral titer and expression profile of Mx-1 (r=-0.15). Mortality records began on day 6. All animals died by day 14. Fish showed typical lesions of IPN disease: a whirling behavior and internal and external signs, such as darkening, pigmentation, mild to moderate exophthalmia, abdominal distention and accumulation of ascites.
Gene expression 16°C: On day 2, ]]>
Fig. 2A) (t-student, p<0.05), and showing a highly significant up-regulation on day 7 (t-student, p<0.001). Additionally, IFN-1(α) expression observed an increase when compared with STAT-1 and Mx-1, except for the last day (Fig. 3C) (t-student, p<0.00001). Furthermore, we observed ]]>
Fig. 2B) (t-student, p<0.05) compared with the value registered at 8ºC, and on day 11 compared with the value registered at 12ºC (t-student, p<0.05). In addition STAT-1 was significantly up-regulated when compared with IFN-1(α) and Mx-1 on day 21 (Fig. 3C) (t-student, p<0.05). We ]]>
Fig. 2C) except on day 11, the time when it showed a similar value with the data detected at 8ºC. Mx-1 was lower during the experiment when compared with the value of IFN-1(α), the latter being highly significant (Fig. 3C) ]]>
Discussion ]]>
The temperature influences biological processes, mainly for fish immune responses (Padron et al., 2000; Raida, & Buchmann, 2007). In this study, we analyzed the effect of temperature changes on the expression of antiviral genes against IPNV.
Moss & Gravell (1969) found ]]>
Lepomis machrochirus) (Saint-Jean et al., 2003). Our results showed viral replication variations in infected fish in day 2 at different tested temperatures. That was caused by the innate response displayed against the virus and not by temperature alterations. It was observed that the virus can replicate at 4°C (Wolf, 1966), although the lowest temperature tested was 8°C.
Since IFN-1(α) activity was discovered for the first time in FMH cells infected with IPNV (Gravell, & Malsberger, 1965), several authors have demonstrated antiviral activity of cytokine and interferon-induced genes (ISGs) (Roberts, & Pearson, 2005; García et al., 2011) against viral infections in teleost. We agreed with the in vitro and
in vivo studies (Kinkelin, & Dorson, 1973; Eaton, 1990; Robertsen, 2006; Verrier, Langevina, Benmansoura, & Boudinota, 2011) which described significant antiviral activity of Mx-1. In our investigation it was evident that at 8°C the viral titer observed was down-regulated by an expression increase of Mx-1 and STAT-1. Additionally, Mx-1 exerted its antiviral activity (Saint-Jean, & Pérez-Prieto, 2007) from the 4th experimental day, possibly interfering with the viral proteins transport (Kochs, Reichelt, Danino, Hinshaw, & Haller, 2005; Wu, Lu, & Chi, 2010). That explains the disappearance of some ]]>
The results obtained at 12°C drew special interests depending on the mortality observed, experimental studies in 60ths (Wolf, 1988) showed high infected salmonids mortality with IPNV at 12°C but still with no explanation. Our possible explanations focus on the implementation of viral strategies for the evasion of the interferon system (Levy, & García, 2001). The first explanation was the significant ]]>
Skjesol et al. (2009) have referred and pointed to VP4 and VP5 proteins like IPNV virulence factors, however is unknown until now. Although, Dobos (1995) found that IPNV ]]>
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The protector effect of IFN-1(α) in fish exposed to 16°C during first day of infection, possibly inhibited viral replication (Kinkelin, & Dorson, 1973). Dios et al. (2010), stimulating larvae of zebra fish (Danio rerio) with Poly-IC observed an inhibition in the expression profile of Mx at 15°C. ]]>
Danio rerio), as a contrary to our work which shows inhibition of Mx at a maximum physiological temperature (16°C). Therefore, the extreme temperature values for each species are critical physiological points to consider for interferon system immune response genes. Our results support that the expression of IFN-1(α), STAT-1 and Mx-1 are physiologically modulated by water temperature, directly influencing ]]>
Acknowledgments
This document was funded by the research project No. 99736 (CONACYT) and would not have been possible ]]>
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1. Autonomous University of the State of Mexico, Faculty of Veterinary Medicine and Animal Husbandry, Advanced Animal Health Research and Study Center, Toluca, AP.4-56, México; darguedas@utn.ac.cr, cortegas@uaemex.mx, simonmc@uaemex.mx
2. Laboratory of Biotechnology and Aquatic Pathology, Austral University of Chile, Valdivia, AP.50-9000, Chile; renrique@uach.cl, alexromero@uach.cl
3. Center INCAR: Interdisciplinary Center for Aquaculture Research, Valdivia, AP.50-9000, Chile; alexromero@uach.cl
4. Laboratorio LARED, Universidad Técnica Nacional, Guanacaste, AP Cañas 60 5700, Costa Rica; darguedas@utn.ac.cr
Received 01-IX-2014. Corrected 10-I-2015. Accepted 11-II-2015.