Diagnosis and genome analysis of lettuce necrotic yellows virus subgroups
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Lettuce necrotic yellows virus is the type species of Cytorhabdovirus. This plant virus causes a disease that is most frequently reported in lettuce in Australia and New Zealand. The lettuce necrotic yellows virus (LNYV) population comprises two subgroups; subgroup I and subgroup II. The subgroups were previously identified by phylogenetic analysis of LNYV, and a diagnostic method distinguishing these subgroups has not yet been developed. In the current study, a diagnostic test for the LNYV subgroups based on reverse transcription polymerase chain reaction (RT-PCR) and RT-PCR- restriction fragment length polymorphism (RFLP) was developed and used for subgroup diagnosis. Subgroup specific primers were designed and tested on known infected samples. RT-PCR diagnosis of LNYV subgroups with these primers requires the subgroup specific primers are used in separate reactions, requiring two reactions for each sample being tested. The RT-PCR-RFLP diagnostic test allows amplification of an LNYV sequence using all the subgroup primers combined, followed by a restriction digest to generate a diagnostic pattern of DNA fragments that can be identified by gel electrophoresis.. The previously designed primers, BCNG1/BCNG2, and LNYV_440F/LNYV_1185R primer pairs were used for LNYV diagnosis. The conditions of these primers were re-optimised for the use in AUT laboratory. The as above mentioned primers were used to test for LNYV and its subgroup on potentially LNYV infected plants collected from Auckland, Waikato and Canterbury. A total of ten samples were tested positive for LNYV; three were subgroup I, six were subgroup II and one sample was LNYV subgroup unknown. LNYV subgroups can now be diagnosed more rapidly than by the previously used sequencing and phylogenetic analysis. The results also showed that LNYV_440F/LNYV_1185R primer pair was more efficient than BCNG1/BCNG2 primers to detect LNYV. The false negative results caused by BCNG1/BCNG2 primer pair could be due to RNA degradation. Only one complete genome of LNYV (subgroup I) has been reported, which was obtained from an Australian isolate. In this study, the complete genomes of LNYV subgroups I and II from New Zealand isolates were sequenced by Illumina HiSeq. Phylogenetic analyses of LNYV genomes and all the available cytorhabdovirus and nucleorhabdovirus genomes were carried out. The results showed that LNYV subgroup I genomes are most closely related to each other than to subgroup II. Lettuce yellow mottle virus was the most closely related to LNYV. Phylogenetic analyses of the LNYV nucleocapsid gene sequences were also performed. The amino acid phylogenetic analysis shows that the AU9 isolate (subgroup II from Australia) appears to be closely related to the common ancestor, which indicates the origin of subgroup II. Since the complete genome or other gene sequences from the AU9 isolate are not available, the origin of LNYV cannot be confirmed. More samples from both Australia and New Zealand are necessary to understand these relationships more clearly. LNYV subgroup I isolate has not been detected in Australia since 1993 and subgroup II may have outcompeted subgroup I in Australia, while this has not occurred in New Zealand. It was hypothesised by previously that subgroup II may have a more efficient relationship with the insect vectors and hosts. The glycoprotein was specifically analysed in the current study because rhabdoviruses use glycoprotein to attach and penetrate to the insect vectors/plant hosts. It was hypothesised that analysis of the glycoprotein may help to determine if subgroup II has a higher efficient relationship with insect/plant hosts than subgroup I. Six characteristics of glycoprotein sequence and 2D structure were analysed. It showed there were differences between the subgroups. However, a 3D structure and mutational analysis are needed to determine if the differences affect its association with the insect/plant hosts.