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A novel monopartite dsRNA virus, tentatively named "sponge gourd amalgavirus 1" (SGAV1), was discovered by high-throughput sequencing in sponge gourd (Luffa cylindrica) displaying mosaic symptoms in Jiashan County, Zhejiang Province, China. The genome of SGAV1 is 3,447 nucleotides in length and contains partially overlapping open reading frames (ORFs) encoding a putative replication factory matrix-like protein and a fusion protein, respectively. The fusion protein of SGAV1 shares 57.07% identity with the homologous protein of salvia miltiorrhiza amalgavirus 1 (accession no. DAZ91057.1). Phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) protein suggests that SGAV1 belongs to the genus Amalgavirus of the family Amalgaviridae. Moreover, analysis of SGAV1-derived small interfering RNAs indicated that SGAV1 was actively replicating in the host plant. Semi-quantitative RT-PCR showed higher levels of SGAV1 expression in leaves than in flowers and fruits. This is the first report of a novel amalgavirus found in sponge gourd in China., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The brown planthopper (BPH), Nilaparvata lugens, is a significant agricultural pest capable of long-distance migration and transmission of viruses that cause severe disease in rice. In this study, we identified a novel segmented RNA virus in a BPH, and this virus exhibited a close relationship to members of a recently discovered virus lineage known as "quenyaviruses" within the viral kingdom Orthornavirae. This newly identified virus was named "Nilaparvata lugens quenyavirus 1" (NLQV1). NLQV1 consists of five positive-sense, single-stranded RNAs, with each segment containing a single open reading frame (ORF). The genomic characteristics and phylogenetic analysis support the classification of NLQV1 as a novel quenyavirus. Notably, all of the genome segments of NLRV contained the 5'-terminal sequence AUCUG. The characteristic virus-derived small interfering RNA (vsiRNA) profile of NLQV1 suggests that the antiviral RNAi pathway of the host BPH was activated in response to virus infection. These findings represent the first documented report of quenyaviruses in planthoppers, contributing to our understanding of quenyaviruses and expanding our knowledge of insect-specific viruses in planthoppers., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Trees and shrubs provide important ecological services. However, few studies have surveyed the virome in trees and shrubs. In this study, we discovered a new positive-sense RNA virus originating from Viburnum odoratissimum, which we named "Vo narna-like virus". The complete genome of Vo narna-like virus is 3,451 nt in length with an open reading frame (ORF) encoding the RNA-dependent RNA polymerase (RdRP) protein. Phylogenetic analysis placed this virus within the betanarnavirus clade, sharing 53.63% amino acid sequence identity with its closest relative, Qingdao RNA virus 2. The complete sequence of the virus was confirmed by rapid amplification of cDNA ends (RACE) and Sanger sequencing. Small interfering RNA (siRNA) analysis indicated that this virus interacts with the RNA interference (RNAi) pathway of V. odoratissimum. This is the first report of a narnavirus in V. odoratissimum., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The complete genomic sequence of a novel robigovirus, provisionally named "Mentha arvensis robigovirus 1" (MARV1), was determined by combining next-generation sequencing (NGS), reverse transcription polymerase chain reaction (RT-PCR), and rapid amplification of cDNA ends (RACE) PCR. The complete genomic sequence of this new virus is 7617 nucleotides in length, excluding the 3' poly(A) tail. The MARV1 genome encodes a putative replicase, "triple gene block" proteins, and a coat protein. Phylogenetic analysis demonstrated that MARV1 is a member of the genus Robigovirus, with closest relationships to African oil palm ringspot virus (AOPRV). Furthermore, MARV1-derived small interfering RNAs (siRNAs) showed typical patterns of plant-virus-derived siRNAs produced by the host antiviral RNA interference pathway. This is the first report of a plant virus of the genus Robigovirus in M. arvensis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Purpose: We aimed to explore the effects of the ciliary sulcus angle (CSA) on accurate prediction of the vault after phakic implantable collamer lens (EVO ICL Model V4c) using the KS formula., Methods: Patients were classified according to the size of CSA: group A, narrow angle (CSA < 30°); group B, normal angle (CSA = 30-90°); and group C, wide angle (CSA > 90°). Further, differences between the actual vault dimensions at 3 months postoperatively and the preoperatively predicted vault dimensions in the three groups were analyzed., Results: This study included 223 eyes of 223 individuals. In groups A-C, the difference in the preoperative vault dimensions of the three groups predicted with the KS formula was not statistically significant (P = 0.056). The actual vault dimensions at 3 months postoperatively were significantly different between the three groups (P < 0.001). Moreover, the difference between the actual and predicted vaults by the KS formula was statistically significant (P < 0.001). In the 3 months, after surgery, the percentages of patients with a low vault (< 250 μm) were 0%, 3%, and 29% in groups A, B, and C, respectively. Further, the percentages of patients with an ideal vault (250-750 μm) in the postoperative period were 66%, 84%, and 71% in groups A, B, and C, respectively. Finally, the percentages of patients with a high vault (> 750 μm) in the postoperative period were 34%, 13%, and 0% in groups A, B, and C, respectively. Notably, the distribution of the vault among the three groups was statistically significant (P < 0.001)., Conclusion: The size of CSA significantly affects the predictiveness of the vault by the KS formula, with the most pronounced effect on the angles < 30° and > 90°. Therefore, CSA should be considered when selecting the lens size using the KS formula preoperatively., Clinical Trial Registration Number: ChiCTR2200065501., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

A novel chuvirus from a southern green stink bug (Nezara viridula) was identified by RNA sequencing in this study and was tentatively named "Ningbo southern green stink bug chuvirus 1" (NBSGSBV-1). The complete genome sequence of NBSGSBV-1 consists of 11,375 nucleotides, and the genome was found to be circular by 'around-the-genome' reverse transcription polymerase chain reaction (RT-PCR) and Sanger sequencing. Three open reading frames (ORFs) were predicted in the NBSGSBV-1 genome, encoding a large polymerase protein (L protein), a glycoprotein (G protein), and a nucleocapsid protein (N protein). A phylogenetic tree was constructed based on all of the currently available RNA-dependent RNA polymerase amino acid sequences of viruses of the family Chuviridae, and NBSGSBV-1 was found to cluster together with Sanya chuvirus 2 and Hubei odonate virus 11, indicating that NBSGSBV-1 might belong to the genus Odonatavirus. Five conserved sites were identified in the L proteins of NBSGSBV-1 and other chuviruses. The abundance and characteristics of the NBSGSBV-1-derived small interfering RNAs suggested that NBSGSBV-1 actively replicates in the host insect. To the best of our knowledge, this is the first report of a chuvirus identified in a member of the insect family Pentatomidae. The discovery and characterization of NBSGSBV-1 will help us to understand the diversity of chuviruses in insects., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The spotted lanternfly (Lycorma delicatula) is an invasive pest that causes serious economic losses in fruit and wood production. Here, we identified a novel iflavirus named "Lycorma delicatula iflavirus 1" (LDIV1), in a spotted lanternfly. The full genome sequence of LDIV1 is 10,222 nt in length and encodes a polyprotein containing a picornavirus capsid-protein-domain-like domain, a cricket paralysis virus capsid superfamily domain, an RNA helicase domain, a peptidase C3 superfamily domain, and an RNA-dependent RNA polymerase (RdRp) domain. LDIV1 replicates in the host insect and activates small interfering RNA (siRNA)-based host antiviral immunity. Phylogenetic analysis demonstrated that LDIV1 is most closely related to an unspecified member of the order Picornavirales, with 61.7% sequence identity in the RdRp region and 57.6% sequence identity in the coat protein region, and thus meets the demarcation criteria for new species in the genus Iflavirus. To the best of our knowledge, LDIV1 is the first virus discovered in L. delicatula., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Arlivirus is currently the only genus in the newly established viral family Lispiviridae. In this study, the complete genome sequence of a novel arlivirus, tentatively named "Nbu stink bug virus 1" (NbuSBV-1), was identified in an individual yellow spotted stink bug, Erthesina fullo (family Pentatomidae, order Hemiptera), which is a widely distributed phytophagous pest in Asia. NbuSBV-1 has a single negative-stranded RNA genome of 13,605 nucleotides in length, and it was predicted to contain six open reading frames (ORFs). Conserved domains of NbuSBV-1 were predicted in ORF1 (a nucleoprotein), ORF4 (a glycoprotein domain), ORF5 (a zinc-finger domain), and ORF6 (an RNA-directed RNA polymerase [RdRP] domain, an mRNA cap domain, and a methyltransferase domain). NbuSBV-1 shares 50.54% amino acid sequence identity in the RdRP region with its closest homolog, Lĭshì spider virus 2. In RdRP-based phylogenetic analysis, NbuSBV-1 was clearly clustered in a clade with other arliviruses. Furthermore, NbuSBV-1-derived small interfering RNAs (siRNAs) showed typical patterns of virus-derived siRNAs produced by the host antiviral RNA interference pathway. As far as we know, NbuSBV-1 is the first arlivirus identified in an insect of the family Pentatomidae., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The ladybird beetle Cheilomenes sexmaculata (family Coccinellidae, order Coleoptera) is a common insect predator of agricultural pests. In this study, the full genome sequence of a novel picorna-like virus, tentatively named "Cheilomenes sexmaculata picorna-like virus 1" (CSPLV1), was identified in C. sexmaculata. The full-length sequence of CSPLV1 is 11,384 nucleotides (nt) in length (excluding the polyA tail), with one predicted open reading frame (ORF) encoding a polyprotein of 3727 amino acids, a 13-nt 5' untranslated region (UTR), and a 187-nt 3' UTR. The ORF of CSPLV1 consists of four distinct domains, including an RNA virus helicase domain (nt 3029-3319), a peptidase domain (nt 5555-6121), an RNA-dependent RNA polymerase domain (nt 7154-8101), and a picorna-like coat protein domain (nt 8606-9283). Phylogenetic analysis based on the conserved RdRP sequence showed that CSPLV1, together with Wuhan house centipede virus 3, Hypera postica associated virus 1, and Diabrotica undecimpunctata virus 1, forms an unclassified group that is closely related to members of the family Solinviviridae. To the best of our knowledge, CSPLV1 is the first picorna-like virus discovered in C. sexmaculata., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Negeviruses are a group of insect-specific viruses that have a wide geographic distribution and broad host range. In recent years, nege-like viruses have been discovered in aphids of various genera of the family Aphididae, including Aphis, Rhopalosiphum, Sitobion, and Indomegoura. Here, we report the complete genome sequence of a nege-like virus isolated from Astegopteryx formosana aphids collected in Guangdong, China, which we have designated as "Astegopteryx formosana nege-like virus" (AFNLV). AFNLV has a genome length of 10,107 nt (excluding the polyA tail) and possesses the typical conserved domains of negeviruses. These include a viral methyltransferase, an S-adenosylmethionine-dependent methyltransferase, a viral helicase, and an RNA-dependent RNA polymerase (RdRP) domain in open reading frame 1 (ORF1), a DiSB-ORF2&#95;chro domain in ORF2, and a SP24 domain in ORF3. The genome of AFNLV shares the highest nucleotide sequence identity (74.89%) with Wuhan house centipede virus, identified in a mixture of barley aphids. As clearly revealed by RdRP-based phylogenetic analysis, AFNLV, together with other negeviruses and nege-like viruses discovered in aphids, formed a distinct "unclassified clade" closely related to members of the proposed genus "Sandewavirus" and the family Kitaviridae. In addition, small interfering RNAs (siRNAs) derived from AFNLV did not exhibit typical characteristics of virus-derived siRNAs processed by the host RNAi-based antiviral pathway. However, the extremely high abundance of viral transcripts (average read coverage 73,403X) strongly suggested that AFNLV might actively replicate in the aphid host. AFNLV described in this study is the first nege-like virus discovered in aphids of the genus Astegopteryx, which will contribute to future study of the co-evolution of nege/nege-like viruses and their host aphids., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The leaf beetle Aulacophora lewisii (family Chrysomelidae, order Coleoptera) is a common insect pest of cucurbitaceous vegetables. In this study, the complete genome sequence of a novel virus from a single leaf beetle was determined using metagenomic sequencing and rapid amplification of cDNA ends. A homology search and phylogenetic analysis suggested that the new virus belongs to the genus Iflavirus, family Iflaviridae, and it was tentatively named "Aulacophora lewisii iflavirus 1" (ALIV1). ALIV1 has a single positive-stranded RNA genome of 9655 nucleotides in length (excluding the polyA tail) that is predicted to encode typical conserved domains of iflaviruses, including two picornavirus-like capsid protein domains, a helicase domain, and an RNA-dependent RNA polymerase (RdRp) domain. Sequence comparisons showed that the full genome sequence of ALIV1 is most similar to that of Brevicoryne brassicae picorna-like virus, with 42.4% sequence identity, and it shares 60% sequence identity in the coat protein region with its closest homolog, Watson virus. The average coverage of the ALIV1 sequence was approximately 5000X, suggesting that it might actively replicate in the host. Phylogenetic analysis based on deduced amino acid sequences suggested that ALIV1 is closely related to Dinocampus coccinellae paralysis virus. To the best of our knowledge, ALIV1 is the first virus discovered in A. lewisii and is also the first iflavirus identified in a member of the genus Aulacophora.

An innovative signal amplification strategy assisted by RNase H is described here for the detection of DNA targets in a universal fashion. A tailor-made RNA molecular beacon (RMB) conjugated with a fluorescence resonance energy transfer (FRET) pair (fluorophore and quencher) was designed, characterized, and combined with the employment of RNase H. Its performance is compared to that of other nucleases including Exonuclease III and T7 exonuclease. Fluorometry, performed best at excitation/emission wavelengths of 490/520 nm, gives an amazingly low detection limit of 23 fM for target DNA. The method was verified by the determination of human hemochromatosis (HFE) gene. It is perceived that the method represents a versatile tool for the detection of a wide range of targets. Graphical Abstract An RNase H-assisted signal amplification (RASA) method for the fluorometric assay of nucleic acids has been developed by using a unique RNA molecular beacon (RNA MB) conjugated with a fluorophore (F) and quencher (Q) pair for signal generation.

The thiophene-modified rhodamine 6G (GYJ) has been synthesized as a novel chemosensor. The sensor has sufficiently high selectivity and sensitivity for the detection of Fe 3+ and Al 3+ ions (M 3+ ) by fluorescence and ultraviolet spectroscopy with a strong ability for anti-interference performance. The binding ratio of M 3+ -GYJ complex was determined to be 2:1 according to the Job's plot. The binding constants for Fe 3+ and Al 3+ were calculated to be 3.91 × 10 8 and 5.26 × 10 8  M -2 , respectively. All these unique features made it particularly favorable for cellular imaging applications. The obvious fluorescence microscopy experiments demonstrated that the probes could contribute to the detection of Fe 3+ and Al 3+ in related cells and biological organs with satisfying resolution. Graphical abstract GYJ has high selectivity and sensitivity for the detection of Fe(III) and Al(III) with the binding ratio of 2:1.

The fijivirus southern rice black-streaked dwarf virus (SRBSDV) causes one of the most serious viral diseases of rice in China and Vietnam. To better understand the molecular basis of SRBSDV infection, a yeast two-hybrid screen of a rice cDNA library was carried out using P8, a minor core protein of SRBSDV, as the bait. A rice Cys2His2-type zinc finger protein (OsZFP) was found to interact with SRBSDV P8. A strong interaction between SRBSDV P8 and OsZFP was then confirmed by pull-down assays, and bimolecular fluorescence complementation assays showed that the in vivo interaction was specifically localized in the nucleus of plant cells. Using a series of deletion mutants, it was shown that both the NTP-binding region of P8 and the first two zinc fingers of OsZFP were crucial for their interaction in plant cells. The localization in the nucleus and activation of transcription in yeast supports the notion that OsZFP is a transcription factor. SRBSDV P8 may play an important role in fijiviral infection and symptom development by interfering with the host transcription activity of OsZFP.

Background: Current guidelines recommend pelvic lymphadenectomy (PLND) for patients with pelvic lymph node metastasis and special state. However, these data and recommendations do not distinguish the role of PLND in different patient groups and confirm the final benefits. The aim of this study was to confirm the efficacy of pelvic lymphadenectomy (PLND) for the different groups of patients., Methods: Data obtained from 7 centers were retrospectively analyzed. Of the patients, 190 pN2-3 penile carcinoma patients confirmed by bilateral inguinal lymph node excision were included in this study. Sixty-nine and 121 of these patients did and did not undergo bilateral PLND, respectively. The baseline differences from the patients were matched by propensity score analysis., Results: In this study, the Kaplan-Meier estimated disease-specific survival (DSS) was not significantly different between the PLND and no-PLND groups (P = 0.796). According to the propensity score matching for T stage, N stage, grade, adjuvant therapies, and lymph node stage (number of inguinal lymph node metastasis and extranodal extension), 48 patients were selected for each group. Among the pN2 patients, the PLND group showed higher DSS rates than the no-surgery group (P = 0.030). However, even after matching, survival did not differ between the PLND and no-PLND patients among all patients (P = 0.609) and pN3 patients (P = 0.417) with comparable DSS., Conclusion: Bilateral PLND may improve survival in pN2 patients. Men with pN3 may not benefit from bilateral PLND.

The genome segment S5 of rice black-streaked dwarf virus (genus Fijivirus, family Reoviridae) is functionally bicistronic in infected plants. It has a conserved second ORF (P5-2) partially overlapping the major ORF in a different reading frame, but its function remains unknown. P5-2 was detected in infected plants, but not in purified viral particles by Western blotting, indicating that it is a non-structural protein. In immunoelectron microscopy, polyclonal antibodies against P5-2 specifically labelled chloroplasts of infected rice plants. When P5-2 fused with green fluorescent protein was transiently expressed in leaves of Nicotiana benthamiana, fluorescence was also co-localized with chloroplasts. Experiments with deletion mutants of P5-2 showed that its N-terminal part was responsible for its targeting to chloroplasts.

P6 of southern rice black-streaked dwarf virus (SRBSDV) is a multifunctional protein that is involved in the formation of viroplasms by interacting with P5-1. Here, we used yeast two-hybrid and bimolecular fluorescence complementation assays to show that there were homologous and heterologous interactions between SRBSDV P6 and P9-1 in yeast and plant cells. Mutational analysis showed that the N-terminal region (residues 1-93) of P6 was necessary for the interaction between P6 and P9-1. Self-interactions only occurred between the full-length P6 or P9-1. P9-1 was able to form viroplasm-like inclusion structures alone in the absence of other viral proteins.

The virus-derived small interfering RNAs (vsiRNAs) of Chinese wheat mosaic virus (CWMV), a member of the genus Furovirus, were characterised from wheat plants by deep sequencing. CWMV vsiRNAs of 21-22 nt in length predominated, suggesting that there might be a conserved mechanism of DCL2 and DCL4 involvement in the biogenesis of vsiRNAs, as well as a common RNA silencing pathway in CWMV-infected wheat plants. The 5'-terminal base of vsiRNAs was biased towards A/U, suggesting that CWMV vsiRNAs might be loaded into diverse AGO-containing RISCs to disturb the gene expression of host plants. Possible targets for some of the vsiRNAs were predicted.

Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus, family Reoviridae. Genome segment S5 has a putative second ORF partially overlapping the major ORF but in a different reading frame. This putative ORF is present in a published sequence and in two Chinese isolates now sequenced. Antibodies were raised against purified P5-1 and P5-2 fusion proteins expressed in a prokaryotic system. In western blots, these antibodies reacted with proteins of about 106 and 27 kDa, respectively, as predicted by sequence analysis. In immunoelectron microscopy, antibodies to P5-1 reacted with viroplasms, indicating that P5-1 is a component of viroplasms, but no labeling was observed with P5-2 antisera. Northern blot assays showed that the genome segment S5 was transcribed as a single mRNA with no subgenomic RNA. These results show that S5 is functionally bicistronic in infected plants. Possible translational mechanisms for P5-2 are discussed.

Southern rice black-streaked dwarf virus (SRBSDV) is a recently described member of the genus Fijivirus, family Reoviridae. The roles of the proteins encoded by the SRBSDV genome have rarely been studied. In a yeast two-hybrid (YTH) assay in which SRBSDV P6, a putatively multifunctional protein, was used as bait and an SRBSDV cDNA library was used as prey, there was a strong interaction between the P6 and P5-1 proteins. The interaction was confirmed by bimolecular fluorescence complement (BiFC) assay in plant cells. YTH analysis using truncated mutants showed that the N-terminal region (amino acids 9-231) of P5-1 is necessary for binding P5-1 to P6 and that the N-terminal fragment (amino acids 1-93) of P6 is necessary for its interaction with P5-1. SRBSDV P5-1 formed granules positioned at the cell periphery in Nicotiana benthamiana leaves; P6 was present in both the cytoplasm and the nucleus and formed punctate bodies associated with the cell periphery. Immunogold labeling showed that both P6 and P5-1 localized within viroplasms in infected cells of rice plants. These results suggest that the interaction between P5-1 and P6 of SRBSDV may be involved in the formation of viroplasms.

Purpose: Metabolic syndrome and insulin resistance have been linked to increased risk of occurrence and mortality of hepatocellular carcinoma (HCC). Recently, retinol-binding protein 4 (RBP4) was clarified as a specific serological marker of insulin resistance. The aim of this study was to determine whether serum RBP4 could be used as a potential marker for predicting prognosis in patients with HCC after curative resection., Methods: Western immunoblotting and Enzyme-linked immunosorbent assay were used to measure the RBP4 expression in cell lines, supernatant, and serum. Serum RBP4 levels were compared with clinicopathological features and outcomes of patients with HCC. Furthermore, we investigated the impact of serum RBP4 level, serum C-peptide level, and HOMA-IR on overall survival (OS) and disease-free survival (DFS) of patients with HCC in the training cohort (156 patients with HCC), and then were validated in the validation cohort (105 patients with HCC)., Results: RBP4 protein overexpressed in HCC cell lines compared with normal liver cell line (P < 0.001) and correlated with metastatic potential. Serum RBP4 levels were associated with OS [hazard ratio (HR) = 2.208, P < 0.001] and DFS (HR = 1.878, P = 0.029) of patients with HCC. By multivariate analysis, the serum RBP4 level was identified as an independent factor for OS (HR = 2.170, P = 0.004) and DFS (HR = 1.769, P = 0.037) of patients with HCC. The prognostic value of serum RBP4 level was confirmed in the validation cohort., Conclusions: The serum RBP4 level is potential to be a useful prognostic factor for HCC after curative resection.

The complete nucleotide sequences of segments S2, S3, S5, and S8-S11 of a Rice gall dwarf virus isolate from Guangxi Province, China, (RGDV-GX) were determined, completing the sequence of this isolate. The total genome (25,567nt) was similar in organization to a recently reported Thailand isolate (RGDV-T). A previously unreported second segment-specific repeat of 7 or 8nt was detected close to the 3'-end of segments S3, S5, and S8. The 3'-UTR of RGDV-GX S4 was 139nt shorter than that of RGDV-T; the insertion in RGDV-T contains a 34-nt inverted repeat, with the 3'-terminus probably abolishing the expected stem loop structure.

The precise role of the poly(A) tail at the 3'-end of the calicivirus RNA genome is unknown. To study the relationship between the presence of the poly(A) tail and the infectivity and replication of rabbit hemorrhagic disease virus (RHDV), mutants of an infectious cDNA clone of RHDV were constructed, and RK13 cells were transfected with transcripts from these mutants. Transcripts with and without a poly(A) had a fairly similar ability to infect and replicate, suggesting that a long 3'-terminal poly(A) is not essential for infectivity and replication. RT-PCR with specific primers, using viral RNA recovered from RK13 cells transfected with poly(A)-deficient RNA transcripts, showed that the poly(A) tail was restored in vivo.

An isolate of a plant reovirus causing severe stunting and dark leaf symptoms on rice from Guangdong, China, was similar in virion morphology and serologically related to rice black-streaked dwarf virus (RBSDV). The electrophoretic profiles of genome segments of the two viruses in agarose or polyacrylamide gel were indistinguishable. The four genome segments of the new isolate corresponding to RBSDV S7-S10 were amplified by ligation RT-PCR and sequenced. The size and organization of each genome segment was very similar to its counterparts in RBSDV, maize rough dwarf virus (MRDV), and mal de Rio Cuarto virus (MRCV). Sequence identity was greatest to RBSDV and MRDV (ranging from about 60-85% depending on the protein), but identities were always much lower than those between RBSDV and MRDV. These comparisons and phylogenetic analyses suggested that the virus represents a new species in genus Fijivirus group 2, tentatively named Rice black-streaked dwarf virus-2.

A filamentous virus isolated from Iris japonica with mosaic symptoms was pathogenic to I. japonica and I. bulleyana but not to 12 virus indicator species. Sequencing of the 3'-terminus of the genome showed that it was a potyvirus, most closely related to sunflower mosaic virus (63.2% identical), and phylogenetic analysis also showed a more distant grouping with tobacco etch and Colombian datura viruses. The virus was detected by RT-PCR in symptomatic I. japonica plants from several regions of China. The virus appears to be an isolate of a new species in the genus Potyvirus, tentatively named butterfly flower mosaic virus.

A potyvirus from Chinese narcissus was transmitted mechanically to three species of Narcissus and to Lycoris radiata but not to 22 other test species. In western blot, the coat protein reacted strongly with Narcissus degeneration virus (UK isolate) antiserum. Antiserum raised to the Chinese virus did not react with eighteen other potyviruses. The complete nucleotide sequence (9816 nt) had the typical genome organisation for a member of the genus Potyvirus. Sequence comparisons and phylogenetic analysis showed that the Chinese virus was different from all previously sequenced potyviruses but distantly related to onion yellow dwarf and shallot yellow stripe viruses.

The '6K1' protein of the Pinellia isolate of Soybean mosaic virus was cloned into a prokaryotic expression vector and a polyclonal antiserum raised to the expressed fusion protein. In immunogold labeling of thin sections of infected leaves of Pinellia ternata, specific labeling occurred at the cell periphery. This might suggest that the potyvirus '6K1' protein plays some role in viral cell-to-cell movement but the lack of transmembrane domains suggests that it does not conform to currently-recognized patterns of viral movement proteins.

Rice gall dwarf virus (RGDV) is a member of the genus Phytoreovirus within the family Reovirdae. Its genome has 12 segments of double-stranded RNA (dsRNA), of which the nucleotide sequences of segments S4, S6, and S7 were determined, providing the first complete genome sequence of RGDV. Each of the segments S4, S6, and S7 contained conserved terminal sequences conforming to the RGDV consensus, 5'-GGXA ... UGAU-3' (X = U or C). Each segment had a single predicted open reading frame encoding proteins with calculated molecular weights of 79.8, 58.6, and 53.3 kDa. These proteins appeared to be homologous to those encoded by the corresponding segments of rice dwarf virus and wound tumor virus, the other known members of the same genus, having about 20-30% amino acid identity to them. It is therefore likely that RGDV S4 and S6 encode non-structural proteins and S7 an inner core protein. Probable homologies between the segments of all known phytoreoviruses are summarized. Beyond these similarities, the RGDV proteins displayed no significant similarity to any other reported viral proteins.

The complete genome sequence of the four RNAs of rice stripe virus Zhejiang isolate was determined. In addition to polymerase modules, the pc1 protein encoded on RNA1 harbours an ovarian tumour (OTU) - like cysteine protease signature near its N-terminus, suggesting that the protein might yield the viral polymerase and one or more additional proteins by autoproteolytic cleavage and/or have deubiquitination activity. A novel inverted repeat sequence motif was found to be universal within the intergenic regions of ambisense genome segments of tenuiviruses, supporting the possibility that it may be functionally important, perhaps in regulating transcription termination.

A filamentous virus, with particles 600-650 nm long, was purified from Narcissus pseudonarcissus (daffodil) in Hangzhou and an antiserum prepared. After mechanical inoculation, the virus could be detected serologically in Narcissus species but not in some commonly used virus indicators. Infection was symptomless. The complete sequence of the genomic RNA (8281 nt) showed six predicted ORFs typical of carlaviruses. Pairwise comparisons of gene sequences and phylogenetic analysis demonstrated that the new virus should be classified as a carlavirus but that it was not closely related to members of any current species. We propose the name Narcissus symptomless virus (NSV).

Infectious cDNA clones of the Shanghai isolate of Ribgrass mosaic virus (RMV) were produced by joining four overlapping cDNA fragments and also in a single step by long template PCR. After inoculation of Nicotiana glutinosa with either RNA transcripts or the cDNA under the control of the CaMV 35S promoter, plants developed typical symptoms, and viral coat protein could be detected in them by Western blot analysis. However, compared to plants inoculated with purified viral RNA, lesions were fewer and appeared more slowly. An epitope of the Mycobacterium tuberculosis 31-kDa protein was inserted at the C-terminus of the viral coat protein by PCR using two overlapping fragments. The modified clone was also infectious and the foreign epitope could be detected serologically in the electron microscope and by Western blot analysis. The results demonstrate the potential of RMV as a viral gene vector.

The complete sequence of an isolate of Narcissus common latent virus (NCLV) from Zhangzhou city, Fujian, China was determined from amplified fragments of purified viral RNA. Excluding the poly(A) tail, the genomic RNA of NCLV was 8539 nucleotides (nt) long and had the typical organization for a member of the genus Carlavirus. The most closely related species were Potato virus M, Hop latent virus and Aconitum latent virus, which had 58-59% nt identity to NCLV in their entire genomes. These relationships were confirmed by a phylogenetic analysis using a composite nucleotide alignment of all the open reading frames.

Plants of Thunberg fritillary (Fritillaria thunbergii Miq.) from Zhejiang Province, were found to be co-infected with two distinct potyviruses. One was an isolate of the recently reported Thunberg fritillary mosaic virus (TFMV; Wei et al., (2005) Arch Virol 150: 1271-1280), while the other was a distinct virus that did not react with TFMV antiserum nor with antisera to 17 other potyviruses, except for a weak reaction with antibodies produced to soybean mosaic virus (SMV) Pinellia strain. Both viruses could be transmitted mechanically to their original host but not to any of a range of commonly used indicator plants. No local lesion host was identified that would enable the viruses to be propagated independently. The complete sequences of both viruses were determined; that of the new virus (9656 nt) had the typical genome organisation and recognised sequence motifs of a potyvirus, encoding a putative polyprotein of 351 kDa. Phylogenetic analysis, sequence comparisons, and the pattern of polyprotein cleavage sites all indicated that it was a member of the Bean common mosaic virus subgroup. The most closely related species are Soybean mosaic virus and Wisteria vein mosaic virus, with 68-69% amino acid identity between their polyproteins. This is sufficiently different for the new virus to be regarded as a distinct species, which we have tentatively named Fritillary virus Y.

The host range and nucleotide sequence of shallot yellow stripe virus (SYSV) from welsh onion in Shandong province, China is described. Of the plants tested, only shallot and welsh onion became infected but most shallot plants were symptomless. The complete sequence of one isolate (10429 nt) and the 3'-terminal 3540 nts of a second isolate were determined. They had c. 90% nt identity to one another and to published (partial) sequences of SYSV. SYSV was most closely related to onion yellow dwarf virus (OYDV) and resembled it in having a much larger P3 protein than other species in the genus.

A potyvirus causing mosaic symptoms in Thunberg fritillary (Fritillaria thunbergii) was found at two sites in Zhejiang province, China. The virus was readily mechanically transmitted to its original host but not to any of 17 other widely used plant virus indicators. A polyclonal antiserum raised to purified virus particles reacted with its homologous virus but not with a range of other viruses (including 16 potyvirus species). In electron microscopy, virus particles and inclusion bodies typical of a potyvirus were seen. The complete nucleotide sequence of an isolate from Ningbo was determined. It was 9723 nt long and sequence analyses predicted the standard potyvirus organisation. The partial sequence (1664 nts at the 3'-terminus) of an isolate from Panan was also determined; the two sequences had 96.9% nt identity. In sequence comparisons and phylogenetic analyses with completely sequenced potyviruses, the new virus was most closely related to Lily mottle virus (53.0% aa identity) and Leek yellow stripe virus. The most closely related incomplete sequence in the international databases was for Lycoris mild mottle virus (72.8% nt identity in their coat proteins). These results suggest that the virus studied is a new species in the genus Potyvirus, which we have tentatively named Thunberg fritillary mosaic virus.

The complete sequence of the genomic RNA of an isolate of Lily virus X (LVX) has been determined for the first time. The isolate from the Netherlands was 5823 nucleotide (nt) long excluding the 3'-poly(A) tail, making it the shortest reported potexvirus sequence. The 5'-non-coding region begins with GGAAAA like that of Scallion virus X (ScaVX) and some isolates of Cymbidium mosaic virus (CymMV), whereas those of other sequenced potexviruses probably all begin with GAAAA. The genome organisation was similar to that of other members of the genus except that a TGBp3-like region lacked a normal AUG start codon. A phylogenetic analysis based on the entire coding sequence showed that LVX was most closely related to Strawberry mild yellow edge virus and belonged in a subgroup of the genus that also contains CymMV, Narcissus mosaic virus, ScaVX, Pepino mosaic virus, Potato aucuba mosaic virus and White clover mosaic virus.

Degenerate primers were used to detect and amplify 3'-terminal genome fragments of potyviruses from medicinal aroid plants growing at 16 sites in China. Virus was detected in 7 samples of which six, all of Pinellia ternata, contained a strain of soybean mosaic virus (SMV) similar to that previously reported from this host in China. The complete sequence of one isolate and the P1 protein coding region of the other isolates were also sequenced. In all cases, the P1 proteins resembled isolates of Dasheen mosaic virus (DsMV) more closely than SMV, confirming earlier suggestions of recombination in this region. In a phylogenetic analysis of SMV, DsMV and related sequences, the aroid sequences of SMV formed a distinct group which also included a sequence published as Zantedeschia symptomless virus (AF469171). One of the P. ternata samples was also infected with a second potyvirus, the 3'-terminal sequence of which was similar to DsMV and to some sequences published as Vanilla mosaic virus. The seventh infected sample was Typhonium flagelliforme and the virus from it was identified from its sequence as zantedeschia mosaic virus (ZaMV), providing the first report of this virus from mainland China.

Tuberose plants with mild mottle symptoms, growing in a glasshouse in Hangzhou, China, contained virions and inclusion bodies typical of a potyvirus. The virus was mechanically transmitted to tuberose but not to 14 other test plant species. A fragment of 4607 nucleotides, corresponding to the 3'-half of a typical potyvirus was amplified by RT-PCR using degenerate primers and sequenced. The most similar sequence in the databases was that of Tuberose mild mosaic virus (TuMMV) from Taiwan and this was the only virus significantly related to it in phylogenetic analyses. The new sequence had 71.1% nt and 76.6% aa identity to TuMMV in the coat protein. Western blot analyses using antisera raised to expressed coat protein showed that the two viruses were serologically related. Although there are no substantial biological data to distinguish the Hangzhou isolate from TuMMV, the molecular difference between the two virus isolates is similar to, or slightly greater than, that between several pairs of well-established potyvirus species. These results therefore suggest that the Hangzhou isolate should be regarded as a new member of the genus Potyvirus, and we have tentatively named it Tuberose mild mottle virus.

Degenerate primers for RT-PCR were designed and used to amplify genome fragments ( c. 750 nt in the coat protein-ORF6 region) of allexiviruses from a total of 28 garlic samples from 24 provinces in China. Many samples contained more than one distinct sequence. A total of 60 different sequences were obtained. Phylogenetic analysis and two-way comparisons were used to assess the status of the sequences and to re-examine the criteria for distinguishing species within the genus. Most of the sequences could be allocated to either Garlic virus D or Garlic virus X on the basis of sequence similarity but some appeared to be intermediate between existing species. There were no sequences of Garlic virus C or Shallot virus X. A comparison with the related genera Carlavirus, Foveavirus and Potexvirus suggests that the published allexivirus species demarcation criteria may have been drawn too tightly and should be re-examined.

A potyvirus isolated from Pinellia ternata in China was characterised and shown to be related to Soybean mosaic virus (SMV). The virus was pathogenic on P. ternata and some soybean cultivars, whereas the local soybean SMV isolate HH5 did not infect P. ternata. Western blot experiments demonstrated a serological relationship between the virus from Pinellia, SMV and Watermelon mosaic virus (WMV). The complete nucleotide sequences of the Pinellia virus (isolate P-1, 9735 nt) and of the Chinese soybean SMV isolates HH5 (9585 nt) and HZ (9588 nt) were determined. A 1733 nt sequence at the 3'-terminus of a second isolate from Pinellia (isolate P-2) was also determined. The predicted polyprotein of isolate P-1 has 83% amino acid (aa) identity with those of published SMV sequences. In many parts of the genome, aa identity was about 90% but it was much lower in the P1 protein region (24-29%), where it more closely resembled Dasheen mosaic virus (62%). The partial sequence of isolate P-2 had 91% nt identity to P-1 and both isolates resembled a recent sequence in the public databases (AF469171) wrongly named Zantedeschia mosaic virus. The two complete SMV soybean sequences had 93-95% nt identity with those of the previously sequenced isolates and >97% amino acid identity. Phylogenetic analysis and comparisons of coat proteins suggest that the Pinellia, WMV and SMV potyviruses should probably be treated as strains of the same species.

Degenerate primers were used to amplify virus sequences from imported lilies in Zhejiang province, China. Two viruses, Lily mottle virus (LMoV, genus Potyvirus) and Lily symptomless virus (LSV, genus Carlavirus) were detected, purified and completely sequenced from a mixed infection in a plant raised from bulbs imported from the Netherlands. The sequence of LMoV was 9644 nt long and encoded a polyprotein of 3095 amino acids with a calculated M(r) of 351.0 kDa that had only 45.1-54.4% identity to other completely sequenced potyviruses. Phylogenetic analysis of the complete polyproteins of members of the genus demonstrated that LMoV was distantly grouped with LYSV, BYMV and ClYVV. Two partial LMoV sequences from different cultivars were identical to one another and very similar (98.3% identical nucleotides) to the corresponding region of the complete sequence. Analysis of the coat protein sequences of LMoV isolates revealed two subgroups, corresponding to the earlier "Tulip breaking virus lily strain" and "Tulip band breaking virus" isolates. Our newly-determined isolates showed an extremely close relationship to the first of these. The LSV sequence was 8393 nucleotides long and had the typical carlavirus genome organization. The ORF1 protein was most closely related to that of Blueberry scorch virus (57.2% identical amino acids). Sequences of 1796 nt at the 3'-end of three additional LSV isolates from different cultivars were very similar (>98% identical nucleotides) to the corresponding region of the complete sequence. This is the first report of complete sequences for LMoV and LSV.

The complete sequence of an isolate of Onion yellow dwarf virus (OYDV) from Yuhang, Zhejiang province, China, was determined. It was 10538 nts in length and was predicted to encode a polyprotein 3403 amino acids (aa) long with a calculated M(r) of 385.1 kDa. The predicted P3 protein (530 aa) was larger than that of any of the potyviruses sequenced to date (344-378 aa). The additional sequence occurs at the N-terminus of the protein, does not represent a duplication from elsewhere in the OYDV genome and could not be matched to any other sequences in the databases. Similar sequences were found in 4 other Chinese OYDV isolates. Phylogenetic analysis of the amino acid sequences of the polyprotein showed that OYDV is distantly related to Pea seed-borne mosaic virus and the potyviruses of grasses and cereals.

Molecular analyses of viruses infecting Chinese scallion (Allium chinense G. Don) showed that the plants did not contain any of the poty-, carla- or allexiviruses that are common in garlic plants in China. The complete sequences of a potyvirus and a potexvirus were determined and these were shown to represent different viruses from any in the databases. They could be transmitted mechanically to scallion but not to other Allium species (including garlic) or to Narcissus. The potyvirus, tentatively named Scallion mosaic virus, has a distant relationship (c. 62% nucleotide identity over the entire genome) to Turnip mosaic virus and Japanese yam mosaic virus, with which it grouped in phylogenetic analyses. Its genome is 9324 nts long, encoding a 341.3 kDa polyprotein of 3001 amino acids. The potexvirus, tentatively named Scallion virus X, has a genome 6987 nts long and its organisation was similar to that of the other potexviruses but with only 46.3-63.2% nucleotides identical to them. It is most closely related to Narcissus mosaic virus but phylogenetic analyses indicate that it should be considered a distinct species. Neither of the viruses have been detected in garlic, although the two host plants are closely related.

The complete nucleotide sequence of a UK isolate of oat mosaic virus (OMV) was determined. RNA1 was 7550 nt long with one large open reading frame potentially encoding a polyprotein of 262.8 kDa with features typical of bymoviruses. RNA2 was 2284 nt in length, substantially smaller than those of other bymoviruses sequenced. It appeared that most of the P2 region had been deleted during repeated mechanical transmission of the isolate. The 3'-UTR of RNA2 was very long (> 1.25 kb) and proved to have a 532 nt slightly overlapping repeat. Phylogenetic analysis confirmed that OMV is an independent member of the genus Bymovirus.

Conserved carla- and potyvirus primers were used in RT-PCR to amplify virus fragments from garlic and other bulb crops in China and the fragments were subsequently sequenced and compared in phylogenetic analyses. Garlic plants from Henan, Hubei, Jiangsu, Shangdong and Yunnan provinces all contained at least one isolate each of Garlic latent virus (genus Carlavirus), Onion yellow dwarf virus (OYDV, genus Potyvirus) and Leek yellow stripe virus (LYSV, genus Potyvirus). The complete sequence of a Zhejiang isolate of LYSV was also determined, providing the first complete sequence of this virus. The genome was 10142 nucleotides long excluding the poly(A) tail and had the typical features of the genus Potyvirus, although some of the amino acids surrounding the polyprotein cleavage sites were unusual. Shallot yellow stripe virus (SYSV) was amplified from bunching onion (Allium fistulosum var. caespitosum) in Zhejiang province, providing the first record of SYSV in China. Lily mottle virus was amplified from dragon-teeth lily (Lilium brownii var. viridulum).

The nucleotide sequences of segments S1 to S6 of a Chinese isolate of Rice black-streaked dwarf virus (RBSDV) were determined. This provides the first complete sequence of a plant pathogenic member of the genus Fijivirus. The complete ten-segment genome has 29,141 nucleotides, making it the largest reovirus genome so far reported. Each of the segments S1-S6 is predicted to encode a single major protein. Protein comparisons indicated that S1 encoded an RNA dependent RNA polymerase, with similarities to that encoded by S1 of Nilaparvata lugens reovirus (NLRV). S2 and S3 appeared to be homologous to S3 and S4 respectively of both Fiji disease virus (FDV) and NLRV. The protein encoded on S4 showed some similarity to that of NLRV S2. The proteins encoded on S5 and S6, though similar in size to those of NLRV S5 and S6, had no detectable homologies to them or to any other known protein.

To assess the potential of an infectious Sindbis virus vector (dsSIN) as a eukaryotic expression vector, dsSIN recombinants which contain each of the rubella virus (RUB) structural proteins (C, E2, and E1) individually were constructed. Expression of the RUB proteins by each dsSIN recombinant was robust and processing was similar to that observed when these proteins were expressed using other vectors. The C and E2 recombinants, each of which contains a cassette of less than 1,000 nts, were stable through low multiplicity amplification; however, the E1 recombinant, which contains a 1700 nt cassette, was not. Therefore, use of the E1 recombinant is restricted to stock derived from the original transfection. The replication rate of dsSIN and the C and E2 recombinants was similar, however, the replication rate of the E1 recombinant was slower. No phenotypic mixing of any of the RUB proteins in recombinant dsSIN virions could be detected.

This study examined the effects of acute administration of delta-9-tetrahydrocannabinol (delta 9-THC), the psychoactive ingredient in marijuana, on extracellular efflux of dopamine (DA) and its metabolites as measured by in vivo microdialysis in nucleus accumbens of conscious, freely-moving rats. delta 9-THC, at low doses (0.5-1.0 mg/kg), which significantly enhance brain stimulation reward (intracranial self-stimulation), significantly increased DA efflux in nucleus accumbens. Augmentation of DA efflux by delta 9-THC was abolished by removal of calcium (Ca++) ions from the perfusion fluid, indicating a Ca(++)-dependence of delta 9-THC's action. Augmentation of DA efflux by delta 9-THC was either totally blocked or significantly attenuated by doses of naloxone as low as 0.1 mg/kg. Given the postulated role of mesocorticolimbic DA circuits in mediating and/or modulating brain stimulation reward, the present data raise the possibility that marijuana's rewarding effects, and hence its euphorigenic effects and abuse potential, may be related to pharmacological augmentation of presynaptic DA mechanisms. Additionally, the DA mechanisms enhanced by marijuana appear to be modulated by an endogenous opioid peptide system.