Norovirus GII.17 Caused Five Outbreaks Linked to Frozen Domestic Bilberries in Finland, 2019.

In March 2019, the Finnish Institute for Health and Welfare and Finnish Food Authority started an outbreak investigation after a notification of food business operators' recall of frozen bilberries due to a norovirus finding. A retrospective search was conducted in the food and waterborne outbreak notification system to identify the notifications linked to norovirus and consumption of bilberries in January-March 2019. Five outbreaks were found in which norovirus GII or GII.17 had been detected in patient samples. A pooled retrospective cohort study was performed for those four in which a questionnaire study had been done. A case was defined as a person with diarrhoea or vomiting within 2 days after consuming a meal studied at one of the outbreak locations. Of 79 participants, 45 (57%) cases were identified. Persons that had consumed foods containing unheated bilberries were three times more likely to get ill than those who had not consumed them (RR 3.1, CI 95% 1.2-8.1, p = 0.02). Norovirus GII.17 was found in 16/17 patient samples sent for further typing. Identical norovirus GII.17 was detected in frozen Finnish bilberries and patient samples. At the berry packaging premises, signs of norovirus GII contamination were found in packaging lines. A new procedure for extracting viral nucleic acid from food and environmental samples was used during the outbreak investigation. Consumption of industrially packed frozen berries as heated would be one of the means to prevent norovirus infections.

Small RNA NGS Revealed the Presence of Cherry Virus A and Little Cherry Virus 1 on Apricots in Hungary.

Fruit trees, such as apricot trees, are constantly exposed to the attack of viruses. As they are propagated in a vegetative way, this risk is present not only in the field, where they remain for decades, but also during their propagation. Metagenomic diagnostic methods, based on next generation sequencing (NGS), offer unique possibilities to reveal all the present pathogens in the investigated sample. Using NGS of small RNAs, a special field of these techniques, we tested leaf samples of different varieties of apricot originating from an isolator house or open field stock nursery. As a result, we identified Cherry virus A (CVA) and little cherry virus 1 (LChV-1) for the first time in Hungary. The NGS results were validated by RT-PCR and also by Northern blot in the case of CVA. Cloned and Sanger sequenced viral-specific PCR products enabled us to investigate their phylogenetic relationships. However, since these pathogens have not been described in our country before, their role in symptom development and modification during co-infection with other viruses requires further investigation.

Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes.

BACKGROUND: Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes. RESULTS: Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1. CONCLUSIONS: Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

Prunus geminivirus A: A Novel Grablovirus Infecting Prunus spp.

Increased use of metagenomics for routine virus diagnosis has led to the characterization of several genus level geminiviruses from tree fruit long thought to exclusively host RNA viruses. In this study, the identification and molecular characterization of a novel geminivirus is reported for the first time in Prunus spp. The virus, provisionally named Prunus geminivirus A (PrGVA), was identified by Illumina sequencing from an asymptomatic plum tree. PrGVA was subsequently confirmed by rolling cycle amplification, cloning, and Sanger sequencing of its complete genome (3,174 to 3,176 nucleotides) from an additional 18 (9 apricot and 9 plum) field isolates. Apart from the nonanucleotide motif TAATATT↓AC present in its virion strand origin of replication, other conserved motifs of PrGVA support its geminiviral origin. PrGVA shared highest complete genome (73 to 74%), coat protein amino acid (83 to 85%) and rep-associated amino acid (74%) identities with Grapevine red blotch virus (GRBV). PrGVA was graft but not mechanically transmissible. Quantitative polymerase chain reaction screening of Prunus spp. in the National Clonal Germplasm Repository collection using newly designed primers and probes revealed 69.4% (apricot), 55.8% (plum), and 8.3% (cherry) incidences of PrGVA. PrGVA is proposed as a novel member of the genus Grablovirus based on its close genome and phylogenetic relationship with GRBV.

New insights into the history of domesticated and wild apricots and its contribution to Plum pox virus resistance.

Studying domesticated species and their wild relatives allows understanding of the mechanisms of population divergence and adaptation, and identifying valuable genetic resources. Apricot is an important fruit in the Northern hemisphere, where it is threatened by the Plum pox virus (PPV), causing the sharka disease. The histories of apricot domestication and of its resistance to sharka are however still poorly understood. We used 18 microsatellite markers to genotype a collection of 230 wild trees from Central Asia and 142 cultivated apricots as representatives of the worldwide cultivated apricot germplasm; we also performed experimental PPV inoculation tests. The genetic markers revealed highest levels of diversity in Central Asian and Chinese wild and cultivated apricots, confirming an origin in this region. In cultivated apricots, Chinese accessions were differentiated from more Western accessions, while cultivated apricots were differentiated from wild apricots. An approximate Bayesian approach indicated that apricots likely underwent two independent domestication events, with bottlenecks, from the same wild population. Central Asian native apricots exhibited genetic subdivision and high frequency of resistance to sharka. Altogether, our results contribute to the understanding of the domestication history of cultivated apricot and point to valuable genetic diversity in the extant genetic resources of wild apricots.

Genome-wide association links candidate genes to resistance to Plum Pox Virus in apricot (Prunus armeniaca).

In fruit tree species, many important traits have been characterized genetically by using single-family descent mapping in progenies segregating for the traits. However, most mapped loci have not been sufficiently resolved to the individual genes due to insufficient progeny sizes for high resolution mapping and the previous lack of whole-genome sequence resources of the study species. To address this problem for Plum Pox Virus (PPV) candidate resistance gene identification in Prunus species, we implemented a genome-wide association (GWA) approach in apricot. This study exploited the broad genetic diversity of the apricot (Prunus armeniaca) germplasm containing resistance to PPV, next-generation sequence-based genotyping, and the high-quality peach (Prunus persica) genome reference sequence for single nucleotide polymorphism (SNP) identification. The results of this GWA study validated previously reported PPV resistance quantitative trait loci (QTL) intervals, highlighted other potential resistance loci, and resolved each to a limited set of candidate genes for further study. This work substantiates the association genetics approach for resolution of QTL to candidate genes in apricot and suggests that this approach could simplify identification of other candidate genes for other marked trait intervals in this germplasm.

Gene Expression Analysis of Plum pox virus (Sharka) Susceptibility/Resistance in Apricot (Prunus armeniaca L.).

RNA-Seq has proven to be a very powerful tool in the analysis of the Plum pox virus (PPV, sharka disease)/Prunus interaction. This technique is an important complementary tool to other means of studying genomics. In this work an analysis of gene expression of resistance/susceptibility to PPV in apricot is performed. RNA-Seq has been applied to analyse the gene expression changes induced by PPV infection in leaves from two full-sib apricot genotypes, "Rojo Pasión" and "Z506-7", resistant and susceptible to PPV, respectively. Transcriptomic analyses revealed the existence of more than 2,000 genes related to the pathogen response and resistance to PPV in apricot. These results showed that the response to infection by the virus in the susceptible genotype is associated with an induction of genes involved in pathogen resistance such as the allene oxide synthase, S-adenosylmethionine synthetase 2 and the major MLP-like protein 423. Over-expression of the Dicer protein 2a may indicate the suppression of a gene silencing mechanism of the plant by PPV HCPro and P1 PPV proteins. On the other hand, there were 164 genes involved in resistance mechanisms that have been identified in apricot, 49 of which are located in the PPVres region (scaffold 1 positions from 8,050,804 to 8,244,925), which is responsible for PPV resistance in apricot. Among these genes in apricot there are several MATH domain-containing genes, although other genes inside (Pleiotropic drug resistance 9 gene) or outside (CAP, Cysteine-rich secretory proteins, Antigen 5 and Pathogenesis-related 1 protein; and LEA, Late embryogenesis abundant protein) PPVres region could also be involved in the resistance.