SJNNV down-regulates RGNNV replication in European sea bass by the induction of the type I interferon system

Abstract

European sea bass is highly susceptible to the betanodavirus RGNNV genotype, although the SJNNV genotype has also been detected in this fish species. The coexistence of both genotypes may affect the replication of both viruses by viral interaction or by stimulation of the host antiviral defense system in which the IFN I system plays a key role. IFN I triggers the transcription of interferon-stimulated genes, including Mx genes, whose expression has been used as a reporter of IFN I activity. The present study evaluated the effect of a primary exposure to an SJNNV isolate on a subsequent RGNNV infection and analyzed the role of the IFN I system in controlling VNNV infections in sea bass using different in vivo approaches. VNNV infection and Mx transcription were comparatively evaluated after single infections, superinfection (SJ+RG) and co-infection (poly I:C+RG). The single RGNNV infection resulted in a 24% survival rate, whereas the previous SJNNV or poly I:C inoculation increased the survival rate up to 96 and 100%, respectively. RGNNV replication in superinfection was reduced compared with RGNNV replication after a single inoculation. Mx transcription analysis shows differential induction of the IFN I system by both isolates. SJNNV was a potent Mx inducer, whereas RGNNV induced lower Mx transcription and did not interfere with the IFN I system triggered by SJNNV or poly I:C. This study demonstrates that an antiviral state exists after SJNNV and poly I:C injection, suggesting that the IFN I system plays an important role against VNNV infections in sea bass.

Figure 1

Kaplan–Meier survival curve, and infective viral particles in fish sampled during challenge 1. A Accumulated survival of European sea bass in different groups within challenge 1. The different letters indicate the significant differences between experimental groups and between challenges 1 and 2 (Figure 4B) (p < 0.05). B RGNNV titers (TCID₅₀/g) in nervous tissue. C SJNNV titers (TCID₅₀/g) in nervous tissue. Titers in fish from the superinfected group (SJ+RG) were calculated after neutralizing the corresponding genotype.

Figure 2

Viral genome in nervous tissue from fish sampled during challenge 1. A RGNNV RNA2 segment copy number. B SJNNV RNA2 segment copy number. Graphics represent the mean values and standard deviation of three samples collected at different time points post-second inoculation from each experimental group. Each sample comprises tissues from three different fish. Different letters indicate the significant differences between groups as well as between samples collected over time within each group (p < 0.05).

Figure 3

Relative Mx transcription in head kidney sampled during challenge 1. A Relative Mx mRNA values after single VNNV inoculation. B Relative Mx mRNA values after the second inoculation. Graphics represent the mean relative values and standard deviation of three independent samples collected from different experimental groups. Different letters indicate significant differences between groups as well as between samples collected throughout time within each group (p < 0.05).

Figure 4

Kinetics of Mx transcription in head kidney after poly I:C inoculation, and Kaplan-Meier survival curve during challenge 2. A Relative Mx transcription in the head kidney at different times after poly I:C inoculation. The graphics represent the mean relative values of Mx mRNA and standard deviation of three individual samples. Different letters indicate significant differences (p < 0.05). B Accumulated survival of European sea bass in different groups within challenge 2. Different letters indicate the significant differences between the experimental groups and between challenge 1 (Figure 1a) and challenge 2 (p < 0.05).

Figure 5

Relative Mx transcription in head kidney sampled during challenge 3. The graphics represent the mean Mx mRNA relative values and standard deviation of three individual samples collected at different times post-inoculation. The letters indicate the significant differences between groups as well as between samples collected throughout time within each group (p < 0.05).