Genome sequence analysis of five Canadian isolates of strawberry mottle virus reveals extensive intra-species diversity and a longer RNA2 with increased coding capacity compared to a previously characterized European isolate

Abstract

In this study, we report the genome sequence of five isolates of strawberry mottle virus (family Secoviridae, order Picornavirales) from strawberry field samples with decline symptoms collected in Eastern Canada. The Canadian isolates differed from the previously characterized European isolate 1134 in that they had a longer RNA2, resulting in a 239-amino-acid extension of the C-terminal region of the polyprotein. Sequence analysis suggests that reassortment and recombination occurred among the isolates. Phylogenetic analysis revealed that the Canadian isolates are diverse, grouping in two separate branches along with isolates from Europe and the Americas.

Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus

“…The complete genomic sequence of SMoV Nova Scotia isolate NSPer3 (accession numbers, KU200456-KU200457">KU200456-KU200457) has been described previously (Bhagwat et al, 2016) and this isolate was the source for all constructs described below. Reverse transcription was conducted using SuperScript IV (Thermo Fisher) and primer P610R (see Table 1 for primers) to generate cDNA which was then used as a template for PCR amplification using Q5 HF DNA polymerase (New England Biolabs).…”

“…Similar to the majority of members of the family Secoviridae (referred to as secovirids), the SMoV genome consists of two positive sense RNA molecules. Each RNA encodes one large polyprotein referred to as P1 (∼215 kDa) and P2 (∼190 kDa) for RNA1 and RNA2, respectively (Thompson et al, 2002; Sanfacon, 2015; Bhagwat et al, 2016). The two polyproteins are presumably cleaved by an RNA1-encoded 3C-like protease (related to the 3C proteases of picornaviruses) (Gorbalenya et al, 1989) to release mature proteins and intermediate precursor proteins made up of two or more protein domains.…”

“…Similar to other secovirids, the SMoV P2 polyprotein contains the domains for a movement protein (MP) and capsid protein (CP) (Sanfacon et al, 2009; Bhagwat et al, 2016). Secovirids encode either one large 55–60 kDa CP (nepoviruses), two with molecular masses of approximately 40 and 20 kDa (comoviruses, fabaviruses, sadwaviruses, and strawberry latent ringspot virus) or three small 20–25 kDa CPs (cheraviruses, torradoviruses, sequiviruses, and waikaviruses) (Sanfacon et al, 2009, 2011).…”

“…For all characterized bipartite secovirids, the MP and CP domains are present in the C-terminal region of P2, with a variable number of protein domains upstream of the MP domain (Sanfacon et al, 2011). However, a region having sequence identity to the two coat protein domains of satsuma dwarf virus (SDV, a related sadwavirus) was found in the central region of the SMoV RNA2 polyprotein rather than at the C-terminus of the polyprotein (Karasev et al, 2001; Thompson et al, 2002; Bhagwat et al, 2016). Interestingly, the C-terminal region of the P2 polyprotein of five Canadian isolates of SMoV is even larger than that previously observed for isolate 1134 from the Netherlands (Bhagwat et al, 2016), with a total coding capacity of approximately 70 kDa downstream of the presumed CP domain.…”

“…An E/G cleavage site was proposed between the MP and CP domains (Thompson et al, 2002). Additional cleavage sites located within the putative CP domain (to delineate two or more possible CPs) or downstream of this domain were not confidently predicted (Thompson et al, 2002; Bhagwat et al, 2016). …”