Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

Abstract

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV‐type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C‐terminal side of the capsid. However, the concurrent mutation in the N‐terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C‐terminal and N‐terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double‐ and triple‐mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C‐terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N‐terminal side is also important for viral replication in vivo.

Nervous necrosis virus viability modulation by water salinity and temperature

“…NNV replication is a temperature‐sensitive process regulated mainly by RNA1 (Hata et al., 2010; Panzarin et al., 2014; Souto et al., 2019). Optimal temperatures for the in vitro growth of the genotypes found in Southern Europe range from 20 to 30°C (Iwamoto et al., 2000; Panzarin et al., 2014), which also seem to correlate with the onset of epizootic outbreaks.…”

Altered conformational structures of nervous necrosis virus surface protrusions and free coat proteins after incubation at moderate-low temperatures

“…The optimum temperature for virus multiplication ranges from 15 °C to 30 °C depending on NNV genotypes and upper limit temperatures range from 32 °C to 35 °C. It has been reported that these temperatures mainly depend on genomic RNA1 which encodes RNA polymerase, although genomic RNA2 coding for CP also plays a role 30–34 . Infectivity of NNV gradually declines at 37 °C.…”

Reassortant viruses threatening fish aquaculture

“…Results revealed that, whether all mutant viruses were infective, mutants showing single mutations (at 245 or 270 amino acid residues, but not both) reduced mortality rates in a 37% or 60% in turbot ( Scophthalmus maximus ) or Senegalese sole, respectively, but highest survival rate was observed in fish infected with the double mutation (at 245 + 270) 63,69,70 . Moreover, in primary cultures of Senegalese sole neurons, all isolates tested (RGNNV, SJNNV and RGNNV/SJNNV) were able to infect and replicate into the cells whilst the recombinant virus with modifications at both 247 and 270 amino acid residues delayed the viral replication, as well as happens in the brain tissue 71,72 . Certainly, the different amino acid residues changes, including an additional shift at residue 20, appear to be involved in different steps of the viral replication such as the adsorption ability and efficiency of viral attachment, viral binding or particles budding 71 .…”

“…63,69,70 Moreover, in primary cultures of Senegalese sole neurons, all isolates tested (RGNNV, SJNNV and RGNNV/SJNNV) were able to infect and replicate into the cells whilst the recombinant virus with modifications at both 247 and 270 amino acid residues delayed the viral replication, as well as happens in the brain tissue. 71,72 Certainly, the different amino acid residues changes, including an additional shift at residue 20, appear to be involved in different steps of the viral replication such as the adsorption ability and efficiency of viral attachment, viral binding or particles budding. 71 Moreover, soles infected with the attenuated mutant (mutation affecting at 245 and 270 residues) exerted down-regulated antiviral immune responses at transcriptional level, 69 demonstrating the crucial role of these residues in the virulence of NNV.…”

“…71,72 Certainly, the different amino acid residues changes, including an additional shift at residue 20, appear to be involved in different steps of the viral replication such as the adsorption ability and efficiency of viral attachment, viral binding or particles budding. 71 Moreover, soles infected with the attenuated mutant (mutation affecting at 245 and 270 residues) exerted down-regulated antiviral immune responses at transcriptional level, 69 demonstrating the crucial role of these residues in the virulence of NNV. Changes at residues 1408 and 1412 of the RNA2, within a predicted steem-loop structure of alphanodavirus essential for its replication, from RGNNV/SJNNV were also evidenced when compared with the reference strains.…”