The first complete genome sequence of narcissus latent virus from Narcissus.

The complete sequence of a narcissus virus isolated from the Netherlands (Narv-NL) was determined to be 8172 nucleotides in length with an open reading frame encoding for 2624 amino acids. Phylogenetic analysis indicates that Narv-NL is clustered with high confidence among representative members from the genus Macluravirus, including artichoke latent virus (ArLV) and Chinese yam necrotic mosaic virus (CYNMV). Sequence analyses indicated Narv-NL shares 67%-69% nucleotide and 51%-68% amino acid sequence identity with ArLV and CYNMV either in the complete ORF or the coat protein (CP) gene, whereas it had 81%-99 % nucleotide and 80%-99 % amino acid sequence identity with the corresponding CP sequences of narcissus latent virus (NLV) isolates, suggesting that Narv-NL is a member of NLV. To our knowledge, this is the first report of the complete sequence of a NLV isolate.

The genetic diversity of narcissus viruses related to turnip mosaic virus blur arbitrary boundaries used to discriminate potyvirus species.

Narcissus plants (Narcissus tazetta var. chinensis) showing mosaic or striping leaves were collected from around Japan, and tested for virus infections using potyvirus-specific primers. Many were found to be infected with a macluravirus and mixtures of different potyviruses, one third of them narcissus yellow stripe virus (NYSV)-like viruses. Genomes of nine of the NYSV-like viruses were sequenced and, together with four already published, provided data for phylogenetic and pairwise identity analyses of their place in the turnip mosaic virus (TuMV) phylogenetic group. Using existing ICTV criteria for defining potyvirus species, the narcissus viruses in TuMV group were found to be from five species; the previously described NLSYV, and four new species we call narcissus virus 1 (NV-1) and narcissus yellow stripe-1 to -3 (NYSV-1, NYSV-2 and NYSV-3). However, as all are from a single host species, and natural recombinants with NV-1 and NYSV-3 'parents have been found in China and India, we also conclude that they could be considered to be members of a single mega-species, narcissus virus; the criteria for defining such a potyvirus species would then be that their polyprotein sequences have greater than 69% identical nucleotides and greater than 75% identical amino acids.

Narcissus yellow stripe virus and Narcissus mosaic virus detection in Narcissus via multiplex TaqMan-based reverse transcription-PCR assay.

AIMS: Development of a multiplex TaqMan RT-qPCR assay to simultaneously detect Narcissus yellow stripe virus (NYSV) and Narcissus mosaic virus (NMV), frequently causing mixed narcissus infection. Feasibility verification was confirmed in natural samples. METHODS AND RESULTS: Primers and probes were designed based on the conserved CP gene regions of NYSV or NMV and their suitability for singleplex and multiplex TaqMan RT-qPCR assays as well as for conventional RT-PCR. Conventional RT-PCR, singleplex and multiplex TaqMan RT-qPCR assays proved to be NYSV and NMV specific. P-values and coefficients of variation of TaqMan RT-qPCR assays indicated high reproducibility. Significantly increased sensitivity was achieved compared to conventional RT-PCR. The detection limit of both viruses was 103 copies with superior correlation coefficients and linear standard curve responses between plasmid concentrations and Ct values. NYSV and NMV infection of narcissus leaves, petals and bulbs could successfully be detected via our multiplex RT-qPCR method at 1·25 mg. CONCLUSION: Our multiplex TaqMan RT-qPCR assay provides rapid, specific, sensitive and reliable testing to simultaneously detect NYSV and NMV, supplying useful routine monitoring for different narcissus samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient identification and discrimination of the narcissus viruses provides reliable information for scientists and conventional growers. Furthermore, it enriches the information of NYSV, NMV and other narcissus viruses.

Evolutionary rates and genetic diversities of mixed potyviruses in Narcissus.

There is no attempt to evaluate evolutionary rates, timescales and diversities of viruses collected from mixedly infected hosts in nature. Plants of the genus Narcissus are a monocotyledon and are susceptible to several viruses. In this study, narcissus plants (Narcissus tazetta var. chinensis) showing mosaic or striping leaves were collected in Japan, and these were investigated for potyvirus infections using potyvirus-specific primers. Individual narcissus plants were found frequently to be mixedly infected with different potyviruses, different isolates and quasispecies of same virus. The viruses were potyviruses and a macluravirus in the family Potyviridae, namely Narcissus late season yellows virus (NLSYV), Narcissus yellow stripe virus (NYSV), Narcissus degeneration virus (NDV), Cyrtanthus elatus virus A (CyEVA) and Narcissus latent virus (NLV). Genetic diversities of coat protein coding region of different virus species were different; NYSV and CyEVA were most diverse whereas NDV was least. Evolutionary rates of all five narcissus viruses were 1.33-7.15×10-3nt/site/year and were similar. The most recent common ancestors (TMRCAs) varied between virus species; NYSV and CyEVA were the oldest whereas NDV was the youngest. Thus, the oldness of TMRCAs of the viruses correlated well with the greatness of nucleotide diversities.

Complete genome analysis of three isolates of narcissus late season yellows virus and two of narcissus yellow stripe virus: three species or one?

Complete genome sequences of two new isolates of narcissus late season yellows virus (NLSYV) from Australia were compared with the other NLSYV genome from China and with two complete genomes of isolates designated narcissus yellow stripe virus (NYSV), one from Australia and the other from China. On the basis of symptoms on natural and experimental host species, and genome sequence identity, the isolates could either be classified as closely related members of three different species or placed together in one taxon. Options for classification of these potyvirus isolates are discussed.

Complete genome sequence of narcissus late season yellows virus infecting Chinese narcissus in China.

The complete genome sequence of a Chinese narcissus isolate of narcissus late season yellows virus from Zhangzhou, China (NLSYV-ZZ), was determined to be 9,651 nucleotides in length, excluding the 3'-terminal poly (A) tail, by amplification and sequencing of virus RNA. The viral genome contains a single long open reading frame of 9,315 nucleotides encoding a polyprotein of 3,105 amino acids. The polyprotein was predicted to be cleaved into ten mature proteins by three viral proteases. Complete genome sequence comparison and phylogenetic analysis indicated that NLSYV-ZZ was most closely related to narcissus yellow stripe virus (NYSV), which was also isolated from narcissus. These viruses shared 69.9 % identity in their complete nucleotide sequences and 77.0 % identity in their polyprotein amino acid sequences.

Complete genome sequences of seven carlavirus and potyvirus isolates from Narcissus and Hippeastrum plants in Australia, and proposals to clarify their naming.

Complete genome sequences were obtained from two isolates of the carlavirus nerine latent virus from hippeastrum and narcissus plants, two isolates of the potyvirus hippeastrum mosaic virus from a hippeastrum plant, and one isolate each of the potyviruses narcissus degeneration virus, narcissus yellow stripe virus and Vallota speciosa virus from narcissus plants. Proposals are made to clarify the current confusion surrounding the naming of some of these viruses.

Colour break in reverse bicolour daffodils is associated with the presence of Narcissus mosaic virus.

BACKGROUND: Daffodils (Narcissus pseudonarcissus) are one of the world's most popular ornamentals. They also provide a scientific model for studying the carotenoid pigments responsible for their yellow and orange flower colours. In reverse bicolour daffodils, the yellow flower trumpet fades to white with age. The flowers of this type of daffodil are particularly prone to colour break whereby, upon opening, the yellow colour of the perianth is observed to be 'broken' into patches of white. This colour break symptom is characteristic of potyviral infections in other ornamentals such as tulips whose colour break is due to alterations in the presence of anthocyanins. However, reverse bicolour flowers displaying colour break show no other virus-like symptoms such as leaf mottling or plant stunting, leading some to argue that the carotenoid-based colour breaking in reverse bicolour flowers may not be caused by virus infection. RESULTS: Although potyviruses have been reported to cause colour break in other flower species, enzyme-linked-immunoassays with an antibody specific to the potyviral family showed that potyviruses were not responsible for the occurrence of colour break in reverse bicolour daffodils. Colour break in this type of daffodil was clearly associated with the presence of large quantities of rod-shaped viral particles of lengths 502-580 nm in tepals. Sap from flowers displaying colour break caused red necrotic lesions on Gomphrena globosa, suggesting the presence of potexvirus. Red necrotic lesions were not observed in this indicator plant when sap from reverse bicolour flowers not showing colour break was used. The reverse transcriptase polymerase reactions using degenerate primers to carla-, potex- and poty-viruses linked viral RNA with colour break and sequencing of the amplified products indicated that the potexvirus Narcissisus mosaic virus was the predominant virus associated with the occurrence of the colour break. CONCLUSIONS: High viral counts were associated with the reverse bicolour daffodil flowers that were displaying colour break but otherwise showed no other symptoms of infection. Narcissus mosaic virus was the virus that was clearly linked to the carotenoid-based colour break.

Narcissus late season yellows virus and Vallota speciosa virus found infecting domestic and wild populations of Narcissus species in Australia.

Isolates of Narcissus late season yellows virus (NLSYV) were identified from domestic and wild Narcissus populations at incidences of 66 and 49%, respectively. NLSYV was also detected in one plant of Clivea miniata. Comparisons of nucleotide and amino acid sequences of the coat protein genes of NLSYV isolates showed that they formed three distinct phylogenetic groups, including one not seen before. Vallota speciosa virus was detected in one domestic population of Narcissus sp. where it infected 70% of the plants. This is the first report of these viruses in Australia, and of NLSYV infecting C. miniata.

Characterisation of an isolate of Narcissus degeneration virus from Chinese narcissus (Narcissus tazetta var. chinensis).

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.

Radial density distribution and symmetry of a Potexvirus, narcissus mosaic virus.

Narcissus mosaic virus is a Potexvirus, a member of the Flexiviridae family of filamentous plant viruses. Fiber diffraction patterns from oriented sols of narcissus mosaic virus have been used to determine the symmetry and structural parameters of the viral helix. The virions have a radius of 55+/-5 A. The viral helix has a pitch of 34.45+/-0.5 A, with 7.8 subunits per turn of the helix. We conclude that all members of the Potexvirus genus have close to 8 subunits per helical turn.

Narcissus symptomless virus: a new carlavirus of daffodils.

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).

Complete nucleotide sequence and affinities of the genomic RNA of Narcissus common latent virus (genus Carlavirus).

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.