Project description:Grapevine line pattern virus (GLPV) was described 30 years ago from Hungary, and in the lack of its sequence until now no additional information about its presence was reported. However High-Throughput Sequencing (HTS) applied on dsRNAs extracts recovered from a grapevine plant (accession Baco22A) infected with GLPV Grapevine line pattern virus (GLPV) allowed us to sequence it with different High-Throughput Sequencing (HTS) methods andthe assembleing of the full genome sequence of this virus. The availability of the sequence allowed us to validate the presence of the virus bot with RT-PCR and with Northern blot hybridization. These methods were also used to test its graft and seed transmission. In accordance as it was originally suggested its genome was found to comprise three RNA segments.Its RNA1 (3.160 bp), RNA2 (2.493 bp) and RNA3 (2.529 bp), encode four proteins, denoted 1a (Methyltransferase, helicase), 2a (RNA-dependent RNA Polymerase), 3a (Movement protein, MP) and 3b (Coat protein, CP). GLPV showed the highest amino acid identity (92%–99%) with all domains of Hop yellow virus (HYV), which is a tentative member of the genus Anulavirus of the family Bromoviridae. The phylogenetic trees constructed based on the amino acid sequences of 2a and 3b also confirmed the belongingness of GLPV to the genus Anulavirus, allocating it in one cluster together with the anulaviruses, and close to HYV. The very high sequence identity found between GLPV and HYV leaves no doubt that both are two isolates of the same viral species.
Project description:As virus diseases cannot be controlled by traditional plant protection methods the risk of their spread have to be minimized on vegetatively propagated plants, such as grapevine. Metagenomics approaches used for virus diagnostics, offer a unique opportunity to reveal the presence of all viral pathogens in the investigated plant, why their usage can reduce the risk of using infected material for a new plantation. Here we used a special field, deep sequencing of virus derived small RNAs, of this high throughput method for virus diagnostics and determined viromes of vineyards in Hungary. With NGS of virus derived small RNAs we could detect not only the viruses tested routinely, but also new ones, which have never been described in Hungary before. Virus presence didn’t correlated with the age of the plantation, moreover phylogenetic analysis of the identified virus isolates suggests that infections mostly caused by the usage of infected propagating material. Our results, validated by other molecular methods, highlighted further questions to be answered before these method can be introduced as a routine, reliable test for grapevine virus diagnostics.
Project description:Arthropod-borne viruses (arboviruses) such as dengue virus (DENV) and Zika virus (ZIKV) pose a significant threat to global health. Novel approaches to control arbovirus spread are focused on harnessing the antiviral immune system of their main vector, the Aedes aegypti mosquito. In arthropods, genes of the Vago family are often presented as analogs of mammalian cytokines with potential antiviral functions, but the role of Vago genes upon virus infection in Ae. aegypti is largely unknown. We performed a phylogenetic analysis of the Vago gene family in Diptera that prompted us to focus on a Vago-like gene that we named VLG-1. Using CRISPR/Cas9-mediated gene editing, we generated a VLG-1 mutant line of Ae. aegypti that revealed a proviral effect of this gene upon DENV and ZIKV infection. In the absence of VLG-1, virus dissemination throughout the mosquito’s body was impaired, albeit not altering virus transmission rates. A tissue-specific transcriptome analysis revealed that the loss of VLG-1 impacted numerous biological processes potentially linked to viral replication, such as the oxidative stress response. Our results challenge the conventional understanding of Vago-like genes as antiviral factors and underscores the need for further research to elucidate the molecular mechanisms underlying mosquito-arbovirus interactions.
Project description:Small RNA libraries were constructed from total RNA from Jasminum sambac plants exhibiting virus-like symptoms. After sequencing, small RNAs were assembled into contigs with MetaVelvet and assembled contigs were aligned against the NR database of NCBI using BLASTx. Top hits that reported a virus as subject were considered putative viral sequences. Based on such alignments, the whole genome of a virus, we tentatively name Jasmine Virus H was recovered and cloned. Two more small RNA libraries were made in a confirmatory experiment. One from Jasminum sambac and another one from Nicotiana benthamiana plants infected with the newly-cloned virus. The small RNA libraries were aligned against the full-length sequence of Jasmine Virus H to determine the spacial distribution of virus-derived small RNAs along the virus genome.