Cloning and Phylogenetic Analysis of Iranian Isolates of Narcissus Latent Virus Based on Sequence of 3´ Region of Genome

Iris spp. is reported to be affected by several viruses in the family Potyviridae including iris mild mosaic virus (IMMV), iris severe mosaic virus (ISMV), iris fulva mosaic virus,  bean yellow mosaic virus (BYMV), turnip mosaic virus (TuMV), ornithogalum mosaic virus (OrMV), narcissus latent virus (NLV), butterfly flower mosaic virus (BFMV), and gladiolus mosaic virus (tentative name). Narcissus latent virus (NLV) is a member of the genus Macluravirus in the family Potyviridae. It has non-enveloped flexuous filamentous virions of 657 nm long and 13 nm wide, which encapsidate a single-stranded, positive-sense RNA molecule of approximately 8,000 nt long. NLV is distributed widely throughout the major planting areas of Japan, New Zealand, and European countries. It is one of the most common viruses infecting narcissus, iris, gladiolus, and nerine, causing significant yield losses and quality deterioration in their bulbs and flowers. Due to the presence of asymptomatic infection of NLV in iris and narcissus, the relevance of its infection in host plants may be severely underrated. As Khorasan Razavi province is one of the major producing areas of ornamental plants in Iran, identification of this virus is a concern. In this study, we attempted to identify NLV infecting iris plants and compare Iranian NLV isolates with other sequences from different geographical regions to provide the first detailed information of phylogenetic characterization of this virus in Iran.

Iris leaf samples showing virus-like symptoms of leaf chlorosis and mosaic were collected from field-grown plants in Khorasan Razavi province. Total RNA was extracted from the field samples using Promega SV Total RNA Isolation Kit (USA). Reverse-transcription polymerase chain reaction (RT-PCR) was performed using specific primer pair CPU-F (5΄-CATTACACCCGACCTGGAACT-3΄) and CPU-R (5΄-CCATTTCAGGGCATTGGAGGA-3΄), which were designed to amplify a 1066 bp fragment of the 3΄-region of NLV genome (encompassing partial NIb (25 nt), complete CP (894 nt), and partial 3'UTR (147 nt)). PCR products and DNA ladder were separated by agarose gel electrophoresis, visualized using DNA Green viewer staining, and photographed with ultraviolet-illumination. Amplified fragments of the expected size were purified, cloned into pTG19-T vector and bi-directionally sequenced. Obtained sequences were phylogenetically compared with the corresponding isolates available in the GenBank after multiple alignments. The phylogenetic tree was constructed based on the nucleotide sequences of the CP-UTR using the neighbor-joining method by MEGA11.

Amplification product (1066 bp) was obtained from five infected samples, but not from healthy samples. The most typical symptoms in positive samples were mosaic, and interveinal chlorosis. Three selected PCR positive samples were cloned into the pTG19-T vector and sequenced. BLASTn analysis of the sequenced data revealed that the PCR-amplified fragments belonged to NLV. Three selected isolates which are referred to as IR, IR2, and IR3 were deposited in GenBank. The previously identified and conserved amino acid sequence motifs described in CP of macluraviruses were present in Iranian CP sequences. The phylogenetic tree placed the NLV sequences into two distinct phylogroups I and II; the Iranian isolates clustered together with isolates from Poland, New Zealand, and United Kingdom into group II. Phylogenetic analysis showed that Iranian isolates shared 77.47 to 98.12% nucleotide sequence identity and 77.70-99.34% amino acid sequence identity with other isolates of NLV. Also, identity of these three isolates in the nucleotide and amino acid levels ranged between 97 to 97.84% and 97.38 to 99.02%, with each other, respectively. Iranian isolates showed the highest nucleotide sequence identity with NLV5_1 isolate (JX270766) from Poland (between 97.65 to 98.12 %) and the lowest with NLV3 isolate (JX270762) from Poland (between 77.47 to 77.95 %).

NLV is a major constraint to iris and narcissus production worldwide. The phylogenetic analysis showed a low correlation between genetic and geographic distances which further emphasizing the importance of the exchange and use of virus-free propagating organs in preventing the dissemination of this virus. It seems that contaminated vegetative organs from some European countries (e.g. Netherlands), which are the major producer and the largest exporters of flowers and ornamentals in the world, can play a significant role in the worldwide distribution of the virus. Identification and the use of more isolates are recommended for a better understanding of the genetic structure and variation of NLV populations on a large geographical scale. The data obtained in this study will be beneficial to improve control strategies for this virus in Iran.