Genome Features of a New Double-Stranded RNA Helper Virus (LBCbarr) from Wine Torulaspora delbrueckii Killer Strains.

The killer phenotype of Torulaspora delbrueckii (Td) and Saccharomyces cerevisiae (Sc) is encoded in the genome of medium-size dsRNA viruses (V-M). Killer strains also contain a helper large size (4.6 kb) dsRNA virus (V-LA) which is required for maintenance and replication of V-M. Another large-size (4.6 kb) dsRNA virus (V-LBC), without known helper activity to date, may join V-LA and V-M in the same yeast. T. delbrueckii Kbarr1 killer strain contains the killer virus Mbarr1 in addition to two L viruses, TdV-LAbarr1 and TdV-LBCbarr1. In contrast, the T. delbrueckii Kbarr2 killer strain contains two M killer viruses (Mbarr1 and M1) and a LBC virus (TdV-LBCbarr2), which has helper capability to maintain both M viruses. The genomes of TdV-LBCbarr1 and TdV-LBCbarr2 were characterized by high-throughput sequencing (HTS). Both RNA genomes share sequence identity and similar organization with their ScV-LBC counterparts. They contain all conserved motifs required for translation, packaging, and replication of viral RNA. Their Gag-Pol amino-acid sequences also contain the features required for cap-snatching and RNA polymerase activity. However, some of these motifs and features are similar to those of LA viruses, which may explain that at least TdV-LBCbarr2 has a helper ability to maintain M killer viruses. Newly sequenced ScV-LBC genomes contained the same motifs and features previously found in LBC viruses, with the same genome location and secondary structure. Sequence comparison showed that LBC viruses belong to two clusters related to each species of yeast. No evidence for associated co-evolution of specific LBC with specific M virus was found. The presence of the same M1 virus in S. cerevisiae and T. delbrueckii raises the possibility of cross-species transmission of M viruses.

Probable Nipa Palm Wine-Associated Hepatitis A Outbreak after Attending a Funeral Ceremony in Sabah.

Foodborne outbreaks of hepatitis A virus (HAV) are most commonly associated with fresh and frozen produce and with various types of shellfish. Alcoholic beverage-borne outbreaks of hepatitis A are extremely rare. Here, we report an outbreak of hepatitis A associated with the consumption of a traditional wine at a funeral ceremony in the Sabah state of Malaysian Borneo. Confirmed cases were determined by serum anti-HAV immunoglobulin M and/or for fecal HAV by reverse transcription polymerase chain reaction (RT-PCR). The amplicons of RT-PCR were subjected to nucleotide sequencing followed by phylogenetic analysis. We conducted a 1:2 case-control study to identify the possible exposure that led to the outbreak. Sixteen patients met the case definition, they were 18 to 58 years old and 90% of them were males. The case-control study showed that the consumption of nipa palm wine during the ceremony was significantly associated (P = 0.0017) with hepatitis A infection (odds ratio, 5.44; 95% CI, 1.80-16.43). Untreated river water was used to dilute the traditional wine, which was assumed to be the source of the infection. Phylogenetically, these viruses belonged to genotype IA and formed an independent cluster with strains from Taiwan, Japan, and the Philippines. This strain might be an emerging HAV in Asian countries. Environmental assessments were performed and environmental samples were negative for HAV. The incidence of hepatitis A in Sabah was also determined and it was 0.795/100,000 population. Strict monitoring of traditional wine production should be implemented by the local authority to prevent future outbreaks.

Identification and Molecular Characterization of Novel Mycoviruses in Saccharomyces and Non-Saccharomyces Yeasts of Oenological Interest.

Wine yeasts can be natural hosts for dsRNA, ssRNA viruses and retrotransposon elements. In this study, high-throughput RNA sequencing combined with bioinformatic analyses unveiled the virome associated to 16 Saccharomyces cerevisiae and 8 non-Saccharomyces strains of oenological interest. Results showed the presence of six viruses and two satellite dsRNAs from four different families, two of which-Partitiviridae and Mitoviridae-were not reported before in yeasts, as well as two ORFan contigs of viral origin. According to phylogenetic analysis, four new putative mycoviruses distributed in Totivirus, Cryspovirus, and Mitovirus genera were identified. The majority of commercial S. cerevisiae strains were confirmed to be the host for helper L-A type totiviruses and satellite M dsRNAs associated with the killer phenotype, both in single and mixed infections with L-BC totiviruses, and two viral sequences belonging to a new cryspovirus putative species discovered here for the first time. Moreover, single infection by a narnavirus 20S-related sequence was also found in one S. cerevisiae strain. Considering the non-Saccharomyces yeasts, Starmerella bacillaris hosted four RNAs of viral origin-two clustering in Totivirus and Mitovirus genera, and two ORFans with putative satellite behavior. This study confirmed the infection of wine yeasts by viruses associated with useful technological characteristics and demonstrated the presence of complex mixed infections with unpredictable biological effects.

The impact of grapevine red blotch disease on Vitis vinifera L. Chardonnay grape and wine composition and sensory attributes over three seasons.

BACKGROUND: Grapevine red blotch virus (GRBV) is a recently discovered DNA virus, which was demonstrated to be responsible for grapevine red blotch disease (GRBD). Its presence has been confirmed in the United States, Canada, Mexico, and South Korea in white and red Vitis vinifera cultivars, including Chardonnay. It has been shown that the three-cornered alfalfa treehopper (Spissistilus festinus) was able to both acquire the GRBV from a grapevine infected and transmit it to healthy grapevines in glasshouse conditions. Studies found that GRBD impacts fruit price, grapevine physiology, and grape berry composition and metabolism in red cultivars. This study evaluated the impact of GRBD on V. vinifera L. Chardonnay grape and wine composition and sensory properties from one vineyard during the 2014, 2015 and 2016 seasons. RESULTS: Grapes from symptomatic red blotch diseased grapevines were lower in total soluble solids, flavan-3-ol, and total phenolic content, and higher in flavonol content when compared to grapes from healthy grapevines. Wines made with grapes from symptomatic grapevines resulted mostly in lower ethanol content and higher pH when compared to wines made from healthy grapevines. Analysis of volatile compounds and descriptive analysis demonstrated that GRBD can impact wine style by altering aroma, flavor, and mouthfeel attributes. CONCLUSIONS: The impacts of GRBD on grape composition directly influenced wine chemistry. The decreased ethanol content impacted not only the levels of volatile compounds but the sensory perception during descriptive analysis. The extent of GRBD impact on the grape composition and wine composition and sensory attributes varied between seasons. © 2019 Society of Chemical Industry.

Survey for major viruses in commercial Vitis vinifera wine grapes in Ontario.

BACKGROUND: In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario. METHODS: A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others. RESULTS: Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2-4 viruses and 4.7% of the samples with 5-6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province. CONCLUSIONS: This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards.

A survey of oenophages during wine making reveals a novel group with unusual genomic characteristics.

Oenophages have so far been mostly isolated from red wines under malolactic fermentation (MLF), and correspond to temperate or ex-temperate phages of Oenococcus oeni. Their genomes are clustered into 4 integrase gene sequence groups, which are also related to the chromosomal integration site. Our aims were to survey the occurrence of oenophages in a broader and more diverse collection of samples than those previously explored. Active phages were isolated from 33 out of 166 samples, which mostly originated from must and MLF. Seventy one phage lysates were produced and 30% were assigned to a novel group with unusual genomic characteristics, called unk. All unk members produced similar RAPD and DNA restriction patterns, were negative by PCR for the signature sequences previously identified in the integrase and endolysin genes of oenophages, and lacked any BamHI restriction site in their genome. The data support that development of additional and novel signature genes for assessing oenophage diversity is now required.

Molecular analysis of the region encoding the lytic system from Oenococcus oeni temperate bacteriophage phi 10MC.

Malolactic fermentation by Oenococcus oeni is a crucial step in wine-making. Oe. oeni phages are thought to be responsible for fermentation failures, yet they have received little attention. After a molecular analysis concerning the phage phi 10MC integration system, this paper focuses on the lytic system. The attP (phage attachment site)-flanking region has been cloned and sequenced. The 1296-bp lysin gene (Lys) was identified in this region. The deduced amino acid sequence showed classical structural features of phage lysins, and this gene product expressed in Escherichia coli had a lytic activity against Oe. oeni. Downstream of Lys, a second ORF was present (P163). According to its amino acid sequence and the location of its gene, the product could be the phi 10MC holin. This study shows that the genomic organization of phage phi 10MC attP-flanking regions is very similar to that of other lactic acid bacteriophages.