Identification of RNA regions that determine temperature sensitivities in betanodaviruses

Abstract

Betanodaviruses, the causative agents of viral nervous necrosis in marine fish, have bipartite positive-sense RNA genomes. The larger genomic segment, RNA1 (~3.1 kb), encodes an RNA-dependent RNA polymerase (protein A), and the smaller genomic segment RNA2 (~1.4 kb) codes for the coat protein. These viruses can be classified into four genotypes, designated striped jack nervous necrosis virus (SJNNV), redspotted grouper nervous necrosis virus (RGNNV), tiger puffer nervous necrosis virus (TPNNV), and barfin flounder nervous necrosis virus (BFNNV), based on similarities in their partial RNA2 sequences. The optimal temperatures for the growth of these viruses are 20-25°C (SJNNV), 25-30°C (RGNNV), 20°C (TPNNV), and 15-20°C (BFNNV). However, little is known about the mechanisms underlying the temperature sensitivity of these viruses. We first constructed two reassortants between SJNNV and RGNNV to test their temperature sensitivity. The levels of viral growth and RNA replication of these reassortants and parental viruses in cultured fish cells were similar at 25°C. However, the levels of all of the viruses but RGNNV were markedly reduced at 30°C. These results indicate that both RNA1 and RNA2 control the temperature sensitivity of betanodaviruses by modulating RNA replication or earlier viral growth processes. We then constructed ten mutated RGNNVs, the RNA1 segments of which were chimeric between SJNNV and RGNNV, and showed that only chimeric viruses bearing the RGNNV RNA1 region, encoding amino acid residues 1-445, grew similarly to the parental RGNNV at 30°C. This portion of protein A is known to serve as a mitochondrial-targeting signal rather than functioning as an enzymatic domain.

In vitro study of the replication capacity of the RGNNV and the SJNNV betanodavirus genotypes and their natural reassortants in response to temperature

“…Remarkably, viruses possessing the polymerase gene of the same genotype exhibited comparable replication trends, and strains with the RNA1 segment of the RGNNV genotype efficiently multiplied at higher temperatures (30 °C). All these data confirm that the RNA1 genetic segment and its encoded protein play a major role in controlling temperature sensitivity of fish nodaviruses, substantiating previous findings by Hata et al [35]. Nevertheless, a possible role of the RNA2 and the RNA3 cannot be ruled out a priori although, to the best of the authors’ knowledge, there is no evidence for the involvement of these molecules in the regulation of betanodavirus replication.…”

“…A number of experimental trials have also demonstrated the effect of temperature, infectious dose and viral multiplication rate on betanodavirus pathogenicity and disease course [36,47-49]. These observations have led to the assumption that betanodavirus replication is most likely a temperature-sensitive process, as previously hypothesized also by Hata et al [35]. With the aim of shedding light into the complex interplay existing between betanodavirus genetic features, environmental conditions and viral replication capacity, the present study investigates the effect of temperature on the in vitro replication of naïve RGNNV and SJNNV strains and on natural reassortants.…”

“…The study has demonstrated that both genetic segments are involved in determining temperature sensitivity of betanodaviruses; however, the RNA1 molecule is capable of regulating this process independently from RNA2, confirming the observation that viral replication is vulnerable to temperature variations [35]. Although the reverse genetics technology provides a suitable experimental model for studying reassortant betanodaviruses, no information is available for natural field strains.…”

“…The polymerase gene of the SJNNV and of the SJNNV/RGNNV viruses was 97.1 and 97.8% identical to that of strain SJNag93 (corresponding to 97.8 and 98.1% amino acid identity). Pairwise similarity was calculated also for the RNA1 nucleotide region spanning from position 84 to position 1419 and putatively responsible for betanodavirus temperature sensitivity [35], which corresponds to the polymerase N-terminal. Nucleotide identity was 96.1% between strains 283.2009 and 367.2.2005 (corresponding to 98.4% amino acid identity) and 97.7% between strains 484.2.2009 and 389/I96 (corresponding to 97.7% amino acid identity).…”

Evidence for and characterization of nervous necrosis virus infection in Pacific cod (Gadus macrocephalus)

“…The possible reason was that the viral polymerase gene of RNA1 evolved significantly more rapidly than the coat protein gene [32]. One possible explanation for this difference in evolutionary rates is that there has been adaptation to local temperature conditions, which is known to modulate viral RNA replication and which is under the control of RNA1 [17]. Interestingly, the present study indicated that this virus strain was also grouped within the cluster of cold-resistant strains according to the results of amino acid alignments of coat protein sequences (Fig.…”