Evidence for a Novel Partitivirus Isolated From Entomopathogenic Nematode Steinernema Ceratophorum


 Nematodes are abundant, yet little is known about their viruses. In this study, we report a novel partitivirus isolated from Entomopathogenic nematode specie “Steinernema ceratophorum, named as Steinernema ceratophorum Partiti-like Virus 1 (ScPV1). The complete genome of ScPV1 is comprised of two dsRNA segments, dsRNA1 (2352 bp) and dsRNA2 (2196 bp) in length. Both dsRNAs contained a single open reading frame (ORF), encoding a putative RNA-dependent RNA polymerase (RdRp) and a coat protein (CP), respectively. The sequences of the RdRp and CP showed the highest similarity (47% and 33% identity, respectively) to Plasmopara viticola associated Partitivirus 7. Multiple sequence alignments and phylogenetic analysis of RdRp of ScPV1 with other selected viruses indicated that ScPV1 is the new member of genus Betapartitivirus in the family Partitiviridae.


Introduction
Nematodes were one group of organisms that were previously thought to be immune to viral infections and has not been adequately explored for infectious viruses. Recent advances in genomic approaches has led to the rapid identi cation of unknown viruses from recalcitrant environments and organisms [2,3]. The rst nematode viruses were detected from Caenorhabditis elegans and C.briggsae [4,5]. Orsay, the rst known infectious virus of nematodes was isolated from C. elegans [4]. In addition, Santeuil and Le Blanc viruses were identi ed from C.briggsae [5]. Sequence analysis of all three nematode viruses revealed that they belong to family Nodaviridae [4,5]. Soon afterwards, rst infectious viruses of plant parasitic nematodes were reported from Heterodera glycines (soybean cyst nematode; SCN) [6,7]. The identi cation of viruses from natural nematode populations supports the notion that nematode viruses might be common but overlooked [4]. EPNs are known to control wide range of insect pests and are safe alternatives to chemical insecticides which are known to be hazardous to environment and human health. [1].
Above evidence indicates that EPNs may also be infected with viruses, in this study, we report a dsRNA virus isolated from Steinernema ceratophorum strain D43. Steinernema ceratophorum Partiti-like Virus 1 belongs to the family Partitiviridae, which is consists of ve genera, Alphapartitivirus, Betapartitivirus, Cryspovirus, Deltapartitivirus, and Gammapartitivirus [8]. Recently, two new genera, "Epsilonpartitivirus" and "Zetapartitivirus", have been proposed in this family [9,10]. Phylogenetic analysis of the RdRp of ScPV1 and other related viruses con rmed that ScPV1 is a new member of the genus Betapartitivirus

Provenance Of Viral Material
The strain D43 was stored at 4℃ refrigerator, was cultivated on the larvae of Galleria mellonella for the purpose of multiplication of D43. Wax moths were incubated at 23℃ for 28 hours. Nematodes were collected in nematode collection dish from dead wax moth placed in dark for 5-7 days and stored at 4℃. The dsRNA was extracted using TransZol RNA extraction kit following the described kit procedures.
Extracted RNA was stored at -80C for the construction of cDNA libraries. The strain D43 was idtin ed by method proposed by Bekal et al [6].
The dsRNA was extracted using CF-11 cellulose (Sigma) chromatography [11]. The contamination of DNA and rRNA was removed by digestion with DNase I and S1 nuclease, respectively, (Takara, Dalian, China), puri ed dsRNAs were electrophoresed in a 1% (w/v) agarose gel. The strain D43 was found to harbor several dsRNA segments. A cDNA library was constructed using the tagged random primer dN6 (5'-GACGTCCAGATCGCGAAT TCNNNNNN-3') and reverse transcriptase [12]. Resulting cDNAs were ampli ed by using a speci c primer set (Forward primer 5'-CCAGGTCATTAGGTTGCTGA-3'/Reverse primer

Sequence Properties
The complete genome of ScPV1 is comprised of two segments here named as dsRNA1 and dsRNA2, are 2352 bp and 2196 bp in length, respectively, with poly (A) tails at their 3'-termini. The 5'-untranslated region (UTR) and 3'-UTR of dsRNA1 were 84 bp and 210 bp long, respectively (Fig. 1A). Whereas 5'-and 3'-UTRs of dsRNA2 were 58 bp and 303 bp long, respectively (Fig. 1A). Alignment of the 5'-and 3'-UTR sequences of both ScPV1 dsRNAs showed that they were conservative each other (Fig. 1B). Sequence analysis revealed that both dsRNAs contain single ORF, ORF1 and ORF2 respectively. ORF1 extends from nt position 85 to 2226, was predicted to encode a 713-aa protein with predicted molecular mass of 83 KDa (Fig. 1A). BLASTp search indicated that dsRNA1 product has the highest similarities with RdRps of Partitiviridae family members particularly with Plasmopara viticola associated Partitivirus 7 (GenBank accession number QHD64796.1; E value = 2e-142, 98% Query coverage and 47% Percent identity). A Conserved Domain Database (CDD) search showed that RdRp contains a conserved viral RdRp domain (RT_like super family, cl02808). Multiple sequence alignment of RdRps based amino acid sequences of ScPV1 and other related viruses indicated six conserved motifs (III-VIII) ( Fig. 2A). ORF2 begins from 59 bp and ends at 1951 bp, encoding a 630-aa protein with calculated molecular mass of 71