Densovirus infection facilitates plant-virus transmission by an aphid.

The interactions among plant viruses, insect vectors, and host plants have been well studied; however, the roles of insect viruses in this system have largely been neglected. We investigated the effects of MpnDV infection on aphid and PVY transmission using bioassays, RNA interference (RNAi), and GC-MS methods and green peach aphid (Myzus persicae (Sulzer)), potato virus Y (PVY), and densovirus (Myzus persicae nicotianae densovirus, MpnDV) as model systems. MpnDV increased the activities of its host, promoting population dispersal and leading to significant proliferation in tobacco plants by significantly enhancing the titer of the sesquiterpene (E)-ß-farnesene (EßF) via up-regulation of expression levels of the MpFPPS1 gene. The proliferation and dispersal of MpnDV-positive individuals were faster than that of MpnDV-negative individuals in PVY-infected tobacco plants, which promoted the transmission of PVY. These results combined showed that an insect virus may facilitate the transmission of a plant virus by enhancing the locomotor activity and population proliferation of insect vectors. These findings provide novel opportunities for controlling insect vectors and plant viruses, which can be used in the development of novel management strategies.

Genomic features of the polyphagous cotton leafworm Spodoptera littoralis.

BACKGROUND: The cotton leafworm, Spodoptera littoralis, is a highly polyphagous pest of many cultivated plants and crops in Africa and Europe. The genome of this pest will help us to further understand the molecular mechanisms of polyphagy. RESULTS: Herein, the high-quality genome of S. littoralis was obtained by Pacific Bioscience (PacBio) sequencing. The assembled genome size of S. littoralis is 436.55 Mb with a scaffold N50 of 6.09 Mb, consisting of 17,207 annotated protein-coding genes. Phylogenetic analysis shows that S. littoralis and its sibling species S. litura diverged about 5.44 million years ago. Expanded gene families were mainly involved in metabolic detoxification and tolerance to toxic xenobiotics based on GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis. Comparative genomics analysis showed that gene families involved in detoxification and chemosensation were significantly expanded in S. littoralis, representing genetic characteristics related to polyphagy and an extensive host range. CONCLUSIONS: We assembled and annotated the reference genome of S. littoralis, and revealed that this pest has the genetic features of strong detoxification capacity, consistent with it being a significant risk to a wide range of host crops. These data resources will provide support for risk assessment and early warning monitoring of major polyphagous agricultural pests.

Novel partiti-like viruses are conditional mutualistic symbionts in their normal lepidopteran host, African armyworm, but parasitic in a novel host, Fall armyworm.

Recent advances in next generation sequencing (NGS) (e.g. metagenomic and transcriptomic sequencing) have facilitated the discovery of a large number of new insect viruses, but the characterization of these viruses is still in its infancy. Here, we report the discovery, using RNA-seq, of three new partiti-like viruses from African armyworm, Spodoptera exempta (Lepidoptera: Noctuidae), which are all vertically-transmitted transovarially from mother to offspring with high efficiency. Experimental studies show that the viruses reduce their host's growth rate and reproduction, but enhance their resistance to a nucleopolyhedrovirus (NPV). Via microinjection, these partiti-like viruses were transinfected into a novel host, a newly-invasive crop pest in sub-Saharan Africa (SSA), the Fall armyworm, S. frugiperda. This revealed that in this new host, these viruses appear to be deleterious without any detectable benefit; reducing their new host's reproductive rate and increasing their susceptibility to NPV. Thus, the partiti-like viruses appear to be conditional mutualistic symbionts in their normal host, S. exempta, but parasitic in the novel host, S. frugiperda. Transcriptome analysis of S. exempta and S. frugiperda infected, or not, with the partiti-like viruses indicates that the viruses may regulate pathways related to immunity and reproduction. These findings suggest a possible pest management strategy via the artificial host-shift of novel viruses discovered by NGS.

Sequencing and phylogenetic characterization of a novel RNA virus in Arma chinensis.

The complete genome of a novel virus from Arma chinensis was determined by RNA sequencing and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10,540 nucleotides (nt) excluding the poly(A) tail. Two non-overlapping open reading frames (ORFs) in the sense direction were predicted: one long ORF at the 5' end of the genome (6,219 nt) that encodes a polypeptide of 2,072 amino acids (aa), and one short ORF at the 3' end of the genome (3,033 nt) that encodes a polypeptide of 1,010 aa. Phylogenetic analysis indicated that the virus clusters within a large cluster of currently unidentified picorna-like viruses with a high bootstrap value. We named the virus isolate Arma chinensis picorna-like virus 1 (AcPV-1). The prevalence of AcPV-1 infection in samples of Arma chinensis from the wild was at a low level (5.48%, 8 positives in 146 samples). Keywords: Arma chinensis; genomic characterization; phylogenetic analysis; Arma chinensis picorna-like virus 1; prevalence.

Sequencing and phylogenetic characterization of a novel Polerovirus from Nicotiana tabacum.

In this study, we reported the complete genome of a novel Polerovirus, named Tobacco yellow virus (TYV), which can be transmitted by Myzus persicae. TYV had a single-stranded RNA genome of 5735 nucleotides in length and contained six putative open reading frames (ORFs). Phylogenetic analysis with whole genome nucleotide sequences and amino acid sequences deduced from the conserved domain of the RNA-dependent RNA polymerase, clustered TYV with Potato leafroll virus from the genus Polerovirus with high bootstrap values. However, TYV clustered with Brassica yellow virus using amino acid sequences deduced from the conserved domain of the coat protein. Taken together with the identities between ORFs in TYV and related ORFs in species from Polerovirus, our results strongly suggested TYV is a novel species of the genus Polerovirus.

Sequencing and phylogenetic characterization of a novel RNA virus genome from Harmonia axyridis.

As a natural predator of many insect pests on its native Asian range, Harmonia axyridis remains amongst the insects whose pathogenic or beneficial microorganisms are yet to be studied. The genome nucleotide (nt) and amino acid sequences of open reading frames (ORFs) of the novel RNA virus were identified. Neighbor-joining (NJ) were constructed using MEGA7 software packages with nt sequences and conserved amino acid sequences of predicted RNA-dependent RNA polymerase (RdRp).The complete genome of a novel virus named Harmonia axyridis virus 1 was determined by RNA-seq and rapid amplification of cDNA ends from H. axyridis, which had a single-stranded RNA genome of 8868 nts in length and contains two putative ORFs. ORF1 encodes a polypeptide of 2182 amino acids, which contained conserved domains for 2 picornavirus-like capsid proteins and one RNA helicase. ORF2 encodes a polypeptide of 655 amino acids, which contained 1 RdRp domain. Phylogenetic analysis of whole genome nt sequences and RdRp deduced amino acid sequences suggested that the virus clustered with several unclassified Hubei picorna-like virus. To our knowledge, this is the first full annotated genome of a novel member of the unclassified group of RNA viruses, infecting H. axyridis in natural field conditions.

Infection of cotton bollworm by Helicoverpa armigera iflavirus decreases larval fitness.

Previously, we reported a novel iflavirus in Helicoverpa armigera (helicoverpa armigera iflavirus, HaIV) and here we report the effects of HaIV on its host. In a laboratory bioassay, HaIV-positive larvae and pupae developed more slowly and had higher mortality than HaIV-negative larvae, suggesting that the virus is pathogenic. The relative fitness of H. armigera decreased with HaIV infection by a ratio of 0.65. Transcriptional analysis indicated that infection significantly changed the expression levels of host genes, with more genes affected at 72 h after inoculation than at 48 h (138 up- and 229 downregulated at 48 h; 185 up- and 299 downregulated at 72 h). Interestingly, pathways related to digestion and absorption were significantly enriched, e.g., protein digestion and absorption, suggesting developmental regulation of the host by HaIV via these pathways. HaIV-infected H. armigera showed significantly downregulated expression of genes encoding cuticular proteins (CPs), essential for structural and protective functions, at 48 h and 72 h, suggesting that HaIV increased larval mortality by downregulating CP gene expression.

Effect of cooling strategies on overall performance of a hybrid personal cooling system incorporated with phase change materials (PCMs) and electric fans.

The effect of four cooling strategies on cooling performance of a hybrid personal cooling system (HPCS) incorporated with phase change materials (PCMs) and electric fans in a hot environment (i.e., Tair = 36 ± 0.5 °C, RH = 59 ± 5%) was investigated. Twelve healthy young male participants underwent four 90-min trials comprising 70 min walking and 20 min resting periods. Cooling strategies adopted in this work were CON (control), PCM-control (PCMs were removed at the end of exercise), Fan-control (fans were switched OFF during the initial 20 min) and PCM&Fan-control (fans were turned ON after 20 min exercising and PCMs were removed after the 70-min exercise). Results demonstrated that the control of electric fans could suppress the mean skin temperature rise to 34.0 °C by over 15 min and also cut down the energy consumption of the HPCS from 15.6 W h to 12.1 W h over the entire 90-min trials. Thus, it is recommended that fans should be turned off at the beginning of hot exposure and switched on once participants felt warm. Our findings also showed that the removal of fully melted PCM packs from the HPCS could enhance the evaporative cooling effect brought about by air circulation. The removal of melted PCMs significantly reduced the physical load by 37.3% and ratings of perceived exertion (RPE) were decreased by 3.5-4.2 RPE units. This could also help quickly restore the PCM energy for future usage. In summary, cooling strategies demonstrated in this work could improve HPCS's overall cooling performance on workers while working in the studied hot environment.

Duplication and expression of horizontally transferred polygalacturonase genes is associated with host range expansion of mirid bugs.

BACKGROUD: Horizontal gene transfer and gene duplication are two major mechanisms contributing to the evolutionary adaptation of organisms. Previously, polygalacturonase genes (PGs) were independently horizontally transferred and underwent multiple duplications in insects (e.g., mirid bugs and beetles). Here, we chose three phytozoophagous mirid bugs (Adelphocoris suturalis, A. fasciaticollis, A. lineolatus) and one zoophytophagous mirid bug (Nesidiocoris tenuis) to detect whether the duplication, molecular evolution, and expression levels of PGs were related to host range expansion in mirid bugs. RESULTS: By RNA-seq, we reported 30, 20, 19 and 8 PGs in A. suturalis, A. fasciaticollis, A. lineolatus and N. tenuis, respectively. Interestingly, the number of PGs was significantly positive correlation to the number of host plants (P = 0.0339) in mirid bugs. Most PGs (> 17) were highly expressed in the three phytozoophagous mirid bugs, while only one PG was relatively highly expressed in the zoophytophagous mirid bug. Natural selection analysis clearly showed that a significant relaxation of selection pressure acted on the PGs in zoophytophagous mirid bugs (K = 0.546, P = 0.0158) rather than in phytozoophagous mirid bugs (K = 1, P = 0.92), suggesting a function constraint of PGs in phytozoophagous mirid bugs. CONCLUSION: Taken together with gene duplication, molecular evolution, and expression levels, our results suggest that PGs are more strictly required by phytozoophagous than by zoophytophagous mirid bugs and that the duplication of PGs is associated with the expansion of host plant ranges in mirid bugs.

Preference of the aphid Myzus persicae (Hemiptera: Aphididae) for tobacco plants at specific stages of potato virus Y infection.

Potato virus Y (PVY) is a common pathogen affecting agricultural production worldwide and is mainly transmitted by Myzus persicae in a non-persistent manner. Insect-borne plant viruses can modify the abundance, performance, and behavior of their vectors by altering host plant features; however, most studies have overlooked the fact that the dynamic progression of virus infection in plants can have variable effects on their vectors. We addressed this point in the present study by dividing the PVY infection process in tobacco into three stages (early state, steady state and late state); delineated by viral copy number. We then compared the differential effects of PVY-infected tobacco (Nicotiana tabacum) plants on the host selection and feeding behavior of M. persicae. We used Y-shaped olfactory apparatus and electrical penetration graph (EPG) methods to evaluate host selection and feeding behavior, respectively. Interestingly, we found that PVY-infected plants at the steady state attracted more aphids than healthy plants, whereas no differences were observed for those at the early and late states. In terms of feeding behavior, intracellular punctures (closely related to PVY acquisition and transmission) were more abundant on PVY-infected tobacco plants at the early and steady states of infection than in uninfected plants. These results indicate that PVY-infected host plants can alter the host selection and feeding behavior of aphids in a stage-dependent manner, which is an important consideration when studying the interactions among host plants, viruses, and insect vectors.

Release and Transformation of BTBPE During the Thermal Treatment of Flame Retardant ABS Plastics.

Thermal scenarios inevitably occur during the lifecycle of engineering plastics laden with brominated flame retardants (BFRs). However, little information on the fate of embedded BFRs during the thermal processes is available. In this study, we measured the release and transformation of a typical BFR, 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), during the thermal treatment of acrylonitrile butadiene styrene (ABS) plastics. The possible thermal scenarios were simulated by varying the heating temperature and atmosphere. The maximum release rate of BTBPE was observed at 350 °C. A release kinetic model was developed to explore the mechanism of BTBPE release while heating ABS. Material-phase diffusion was found to be the rate-determining step during release. According to the developed release model, it was estimated that 0.04-0.17% of embedded BTBPE could be released to air during the industrial processing of ABS plastics. When the heating temperature was ≥350 °C, approximately 15-56% of embedded BTBPE decomposed to bromophenols (BPs) and 1,3,5-tribromo-2-(vinyloxy) benzene (TBVOB), and the decomposition followed a first-order kinetics at 350 °C. Polybrominated dibenzo- p-dioxins and dibenzofurans (PBDD/Fs) were also significantly formed at ≥350 °C from BPs and TBVOB via a precursor mechanism. A higher temperature (≥450 °C) was favorable for the formation of PBDFs.

Discovery and characterization of a novel picorna-like RNA virus in the cotton bollworm Helicoverpa armigera.

We characterize a novel picorna-like virus, named Helicoverpa armigera Nora virus (HaNV), with a genome length of 11,200 nts, the sequence of which was isolated from the lepidopteran host cotton bollworm Helicoverpa armigera, using RNA-Seq. Phylogenetic analysis, using the putative amino acid sequence of the conserved RNA-dependent RNA polymerase (RdRp) domain, indicated that HaNV clustered with Spodoptera exigua Nora virus, Drosophila Nora virus and Nasonia vitripennis virus-3 with a high bootstrap value (100%), which might indicate a new viral family within the order Picornavirales. HaNV was efficiently horizontally transmitted between hosts via contaminated food, and transmission was found to be dose-dependent (up to 100% efficiency with 109 viral copy number/µl). HaNV was also found to be transmitted vertically from parent to offspring, mainly through transovum transmission (virus contamination on the surface of the eggs), but having a lower transmission efficiency (around 43%). Infection distribution within the host was also investigated, with HaNV mainly found in only the gut of both adult moths and larvae (>90%). Moreover, our results showed that HaNV appears not to be an overtly pathogenic virus to its host.

Characterization of UDP-Glucuronosyltransferases and the Potential Contribution to Nicotine Tolerance in Myzus persicae.

Uridine diphosphate (UDP)-glycosyltransferases (UGTs) are major phase II detoxification enzymes involved in glycosylation of lipophilic endobiotics and xenobiotics, including phytoalexins. Nicotine, one of the most abundant secondary plant metabolites in tobacco, is highly toxic to herbivorous insects. Plant-herbivore competition is the major impetus for the evolution of large superfamilies of UGTs and other detoxification enzymes. However, UGT functions in green peach aphid (Myzus persicae) adaptation are unknown. In this study, we show that UGT inhibitors (sulfinpyrazone and 5-nitrouracil) significantly increased nicotine toxicity in M. persicae nicotianae, suggesting that UGTs may be involved in nicotine tolerance. In total, 101 UGT transcripts identified in the M. persicae genome/transcriptome were renamed according to the UGT Nomenclature Committee guidelines and grouped into 11 families, UGT329, UGT330, UGT339, UGT341-UGT345, and UGT348-UGT350, with UGT344 containing the most (57). Ten UGTs (UGT330A3, UGT339A2, UGT341A6, UGT342B3, UGT343C3, UGT344D5, UGT344D8, UGT348A3, UGT349A3, and UGT350A3) were highly expressed in M. persicae nicotianae compared to M. persicae sensu stricto. Knockdown of four UGTs (UGT330A3, UGT344D5, UGT348A3, and UGT349A3) significantly increased M. persicae nicotianae sensitivity to nicotine, suggesting that UGT expression in this subspecies may be associated with nicotine tolerance and thus host adaptation. This study reveals possible UGTs relevant to nicotine adaptation in tobacco-consuming M. persicae nicotianae, and the findings will facilitate further validation of the roles of these UGTs in nicotine tolerance.

Transcription Factors AhR/ARNT Regulate the Expression of CYP6CY3 and CYP6CY4 Switch Conferring Nicotine Adaptation.

Nicotine is one of the most toxic secondary plant metabolites in nature and it is highly toxic to herbivorous insects. The overexpression of CYP6CY3 and its homologous isozyme CYP6CY4 in Myzus persicae nicotianae is correlated with nicotine tolerance. The expanded (AC)n repeat in promoter is the cis element for CYP6CY3 transcription. These repeat sequences are conserved in the CYP6CY3 gene from Aphis gossypii and the homologous P450 genes in Acyrthosiphon pisum. The potential transcriptional factors that may regulate CYP6CY3 were isolated by DNA pulldown and sequenced in order to investigate the underlying transcriptional regulation mechanism of CYP6CY3. These identified transcriptional factors, AhR and ARNT, whose abundance was highly correlated with an abundance of the CYP6CY3 gene, were validated. RNAi and co-transfection results further confirm that AhR and ARNT play a major role in the transcriptional regulation of the CYP6CY3 gene. When the CYP6CY3 transcript is destabilized by AhR/ARNT RNAi, the transcription of the CYP6CY4 is dramatically up-regulated, indicating a compensatory mechanism between the CYP6CY3 and CYP6CY4 genes. Our present study sheds light on the CYP6CY3 and CYP6CY4 mediated nicotine adaption of M. persicae nicotianae to tobacco. The current studies shed light on the molecular mechanisms that underlie the genotypic and phenotypic changes that are involved in insect host shifts and we conclude that AhR/ARNT regulate the expression of CYP6CY3 and CYP6CY4 cooperatively, conferring the nicotine adaption of M. persicae nicotianae to tobacco.

Performance enhancement of hybrid personal cooling clothing in a hot environment: PCM cooling energy management with additional insulation.

A novel design of personal cooling clothing incorporating additional insulation sandwiched between phase change materials (PCMs) and clothing outer layer is proposed. Performance of four personal cooling systems including clothing with only PCMs, clothing with PCMs and insulation (PCM + INS), clothing with PCMs and ventilation fans (HYB), and clothing with PCMs, ventilation fans and insulation (HYB + INS) was investigated. Effect of additional insulation on clothing cooling performance in terms of human physiological and perceptual responses was also examined. Human trials were carried out in a hot environment (i.e. 36 °C, RH = 59%). Results showed that significantly lower mean skin/torso temperatures were registered in HYB + INS as compared to HYB. In contrast, no significant effect of the use of insulation on both skin and body temperatures between PCM and PCM + INS was observed. Also, no significant difference in thermal sensations, thermal comfort, and skin wetness sensation was registered between cooling systems with and without additional insulation. Practitioner Summary: Hybrid personal cooling clothing has shown the ability to provide a relatively cool microclimate around the wearer' body while working in hot environments. The present work addresses the importance of cooling energy saving for PCMs in a hot environment. This work contributes to optimising cooling performance of hybrid personal cooling systems.

Characterization of a novel RNA virus from Nesidiocoris tenuis related to members of the genus Iflavirus.

The complete genome of a novel virus from Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends. This virus has a single-stranded RNA genome of 10633 nucleotides (nt) in length, not including the poly(A) tail, and contains two putative open reading frames (ORFs). ORF1 encodes a polypeptide of 1320 amino acids (aa) with a predicted molecular mass of 147.92 kDa and theoretical isoelectric point (pI) of 6.96. ORF2 encodes a polypeptide of 1728 aa with a predicted molecular mass of 197.09 kDa and pI of 6.73. Phylogenetic analysis with the deduced aa sequences of the conserved RNA dependent RNA polymerase domain as well as whole genome nt sequences indicated that the virus clusters with viruses classified within the genus Iflavirus, with a high bootstrap value in the maximum-likelihood and neighbor-joining trees. However, this virus has a distinct genome structure with two ORFs, iflaviruses normally having one, suggesting the virus might be a prototype of a new genus. We named the virus isolate Nesidiocoris tenuis virus 1 (NtV-1). The prevalence of NtV-1 infection in wild samples of N. tenuis was at a low level (7.32%, 6 positive in 82 samples), suggesting a possible harmful effect to its host.

Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells.

BACKGROUND: Densoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further. RESULTS: Phylogenetic analysis showed that HaDV2 was similar to members of the genus Iteradensovirus. However, compared to current members of the genus Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the nucleotide-level, and lower than 36, 28 and 19% at the amino-acid-level of VP, NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 were smaller than those from other iteradensoviruses due to their shorter N-terminal sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP proteins were identified by Northern Blot and RACE analysis. Using specific anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins within the cell nucleus was determined by using Green Fluorescent Protein (GFP) labelling. CONCLUSION: The genome organization, terminal hairpin structure, transcription and expression strategies as well as the mutualistic relationship with its host, suggested that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily Densovirinae.

Structural proteins of Helicoverpa armigera densovirus 2 enhance transcription of viral genes through transactivation.

Herein, we report the identification of putative promoters for the non-structural proteins (NS) and capsid structural proteins (VP) of Helicoverpa armigera densovirus (HaDV2) as well as a potential mechanism for how these promoters might be regulated. For the first time, we report that VP is able to transactivate the VP promoter and, to a lesser degree, the NS promoter in densoviruses. In addition to this, another promoter-like sequence designated P2, when co-transfected with the VP gene, enhanced luciferase activity by approximately 35 times compared to a control. This suggests that there are two promoters for VP in HaDV2 and that the VP of parvoviruses might play a more important role in viral transcription than previously appreciated.

The genome sequence of a novel RNA virus in Adelphocoris suturalis.

The complete genome of a novel virus found in Adelphocoris suturalis was determined by RNA-seq and named Adelphocoris suturalis-associated virus 1 (ASV1). ASV1 has a single-stranded RNA genome of 10,845 nucleotides in length and contains five putative open reading frames (ORFs). ORF1 encodes a polypeptide of 2592 amino acids (aa) and contains four conserved domains: a viral RNA methyltransferase domain, an FtsJ-like methyltransferase domain, a viral RNA helicase domain and an RNA-dependent RNA polymerase domain. ORF2, ORF3, ORF4 and ORF5 encode polypeptides of 190, 461, 103 and 159 aa, respectively, of which only ORF5 contains a conserved domain, the Tobacco mosaic virus-coat superfamily. Phylogenetic analysis with the deduced amino acid sequences indicated that ASV1 clusters with the Drosophila-related Boutonnet virus. The similar genomic structure and high bootstrap value identified in the maximum-likelihood tree suggest that ASV1 (possibly alongside Boutonnet virus) could be considered the prototype of a new taxon of unclassified insect viruses. The prevalence of ASV1 infection in wild populations of A. suturalis was at a low level (6.60%, 14 positives from 212 samples).

A novel single-stranded RNA virus in Nesidiocoris tenuis.

The complete genome sequence of a novel single-stranded RNA virus in Nesidiocoris tenuis was determined by RNA-seq and rapid amplification of cDNA ends (RACE) methodologies and was named N. tenuis virus 1. The genomic RNA was 3970 nucleotides (nt) in length and contained two putative open reading frames (ORFs). ORF1 encoded a polypeptide with 283 amino acids containing a viral (superfamily 1) RNA helicase (Hel) domain, and ORF2 encoded a polypeptide with 294 amino acids containing an RNA-dependent RNA polymerase (RdRP) domain. Phylogenetic analysis using the deduced amino acid sequences indicated that the N. tenuis virus 1 clustered with Blackford virus; however, the low bootstrap values and unique genomic structure suggested that the virus is a prototype of a new type of unclassified viruses. The prevalence of N. tenuis virus 1 infection in field populations of N. tenuis differed between three locations, with 28.32% of the 113 sampled individuals testing positive for the virus.