Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence

African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.

Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.

Case Report: Diabetes in Chinese Bloom Syndrome

Bloom syndrome (BS) is a rare autosomal recessive disorder that causes several endocrine abnormalities. So far, only one BS pedigree, without diabetes, has been reported in the Chinese population. We presented the first case of BS with diabetes in the Chinese population and explored the clinical spectrum associated with endocrine. Possible molecular mechanisms were also investigated. Our study indicated that BS may be one rare cause of diabetes in the Chinese population. We also found a new pathogenic sequence variant in BLM (BLM RecQ like helicase gene)(NM_000057.4) c.692T>G, which may expand the spectrum of BLM variants.

An unusually long 5’UTR of ATP dependent cold shock DEAD-box RNA helicase gene csdA negatively regulates its own expression in Escherichia coli

Cold-shock DEAD-box protein A (CsdA) is an ATP dependant cold shock DEAD-box RNA helicase. It is a major cold shock protein needed for the cold adaptation in Escherichia coli. Although the CsdA has been studied at the protein level, further studies are necessary to understand its mechanisms of gene regulations. In this regard, we have constructed a promoter less vector with the ORF of a GFP reporter and found that the promoter of the csdA gene resides far upstream (more than 800 bases) of its coding region. Furthermore, our in vivo deletion experiment has confirmed the existence of this extraordinarily long 5’UTR. Our results show that it represses its own expression. In addition, the short peptide encoding (26 aa) yrbN gene resides within this 5’UTR as an operon with 8 overlapping nucleotides with the csdA coding region. Besides, we observed that the csdA gene expression may also occur along with immediate upstream (180 nucleotides) nlpI gene both at 37°C and 15°C and from the pnp gene (1173 nucleotides upstream) only during cold. In conclusion, csdA gene has operon feature like prokaryotes, in contrast, it also contains an extraordinarily long 5’UTR, found in eukaryotes.

First report of Raspberry leaf mottle virus infecting raspberry in Poland

Raspberry leaf mottle virus (RLMV) in Europe has been associated with raspberry mosaic disease (Martin et al. 2013) and crumbly fruit disease (Quito-Avila et al. 2014) in red raspberry (Rubus idaeus L.) beside Rubus yellow net virus (RYNV) and Black raspberry necrosis virus (BRNV). RLMV has also been reported infecting blackberry (Jones and McGavin 1998; Thekke-Veetil et al. 2016). A survey of virus diseases was conducted from 2016 to 2018 in 34 raspberry commercial plantations in Central and southeastern Poland. Some of the plants showed chlorotic spots, vein yellows, mosaic patterns and deformation of leaves and fruits. Leaf samples from 146 plants were tested using RT-PCR for RLMV, RYNV, BRNV, Raspberry vein chlorosis virus (RVCV), Raspberry leaf blotch virus (RLBV), and Raspberry bushy dwarf virus (RBDV). The pairs of primers CPhF and CPhR (Tzanetakis et al. 2007) as well as 1436F and 1437R (McGavin and MacFarlane 2010) were used to amplify a 452-bp fragment of the coat protein homologue (CPh) gene and a 681-bp of the 1 a helicase gene, respectively. RT-PCR products were obtained for five samples from cultivars: Malling Promise (MPrOK1 and MPrOk2; plantation A), Canby (CanKop; plantation B), Polka (PolKop; plantation B) and Malling Seedling (MSeWa; plantation C) grown in southeastern Poland. MPrOK1 and MSeWa were co-infected with RYNV while the other three samples were mixed infected with RBDV. Each of these plants showed chlorotic spots and mosaic symptoms of different intensity, leaf deformation and reduced growth. The RLMV-specific amplicons obtained for all five positive samples were directly sequenced at Genomed company, Warsaw, Poland. The obtained sequences were aligned using ClustalW of the Lasergene 7.1 software (DNASTAR Inc., USA) and were compared with sequences available in GenBank using the BLASTn algorithm. The identity of nucleotide (nt) sequences of Cph gene of RLMV isolates found in Poland was revealed. They shared 97.7% and 99% nt sequence similarity with ‘HCRL Glen Clova’ from USA (DQ357218) and ‘SCRI stock’ isolate from United Kingdom (FN391551) strains respectively The nucleotide sequence of isolates from Poland indicates a closer relationship with the strain found in UK. However, the amino acid (aa) sequence of both reference strains was identical and shared 99.2% identity with the corresponding fragment of the isolates from Poland. The representative sequence of PolKop isolate was deposited in GenBank under accession No. MT241265. The similarity of nucleotide sequence of the 1 a helicase gene ranged from 98.5% (between MseWa and four other isolates) to 100% (between MPrOk1 and MPrOk2). The amino acids sequences of MPrOk1, MPrOk2 and MSeWa isolates were identical whereas they shared 99.5% similarity with CanKop and PolKop isolates. The nt and aa sequences within this region of all five isolates were in 98% homologous with the sequences of ‘HCRL Glen Clova’ strain. Nucleotide sequences of the 1 a helicase gene of the CanKop, PolKop, MseWa, MPrOk1, MPrOk2 isolates were deposited in GenBank (acc. Nos. MT847633–MT8347637). Considering the low incidence of RLMV (five out of 146 positively tested), it can be assumed that there is no emergency of the RLMV large-scale occurrence in Poland. However, as RLMV is transmitted by Amphorophora idaei aphids found in our country, infected plants near raspberry plantations may play an important role in virus spread. This is the first report of RLMV infecting red raspberry in Poland. Acknowledgments This work was performed in the frame of multiannual programme 2015-2020 "Actions to improve the competitiveness and innovation in the horticultural sector with regard to quality and food safety and environmental protection” (task 2.1), financed by the Polish Ministry of Agriculture and Rural Development. I would like to thank Mrs. Dorota Starzec for technical assistance. References Jones, A. T, and McGavin, W. J. 1998. Ann. Appl. Biol. 132:239. https://doi.org/10.1111/j.1744-7348.1998.tb05200.x. Martin, R. R. et al. 2013. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-04-12-0362-FE. McGavin, W. J. and MacFarlane, S. A. 2010. Ann. Appl. Biol. 156:439. doi:10.1111/j.1744-7348.2010.00401.x. Quito-Avila, D. F., et al. 2014. Plant Dis. 98:176. http://dx.doi.org/10.1094/PDIS-05-13-0562-RE. Thekke-Veetil, T., et al. 2016. Plant Dis. 101:265. https://doi.org/10.1094/PDIS-07-16-1014-PDN Tzanetakis, I. E., et al. 2007. Virus Res. 127:26. doi:10.1016/j.virusres.2007.03.010.

Investigation of a Novel Salt Stress-Responsive Pathway Mediated by Arabidopsis DEAD-Box RNA Helicase Gene AtRH17 Using RNA-Seq Analysis

Previously, we reported that overexpression of AtRH17, an Arabidopsis DEAD-box RNA helicase gene, confers salt stress-tolerance via a pathway other than the well-known salt stress-responsive pathways. To decipher the salt stress-responsive pathway in AtRH17-overexpressing transgenic plants (OXs), we performed RNA-Sequencing and identified 397 differentially expressed genes between wild type (WT) and AtRH17 OXs. Among them, 286 genes were upregulated and 111 genes were downregulated in AtRH17 OXs relative to WT. Gene ontology annotation enrichment and KEGG pathway analysis showed that the 397 upregulated and downregulated genes are involved in various biological functions including secretion, signaling, detoxification, metabolic pathways, catabolic pathways, and biosynthesis of secondary metabolites as well as in stress responses. Genevestigator analysis of the upregulated genes showed that nine genes, namely, LEA4-5, GSTF6, DIN2/BGLU30, TSPO, GSTF7, LEA18, HAI1, ABR, and LTI30, were upregulated in Arabidopsis under salt, osmotic, and drought stress conditions. In particular, the expression levels of LEA4-5, TSPO, and ABR were higher in AtRH17 OXs than in WT under salt stress condition. Taken together, our results suggest that a high AtRH17 expression confers salt stress-tolerance through a novel salt stress-responsive pathway involving nine genes, other than the well-known ABA-dependent and ABA-independent pathways.