Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Identification of Pistacia-associated flexivirus 1, a putative mycovirus of the family Gammaflexiviridae, in the mastic tree (Pistacia lentiscus) transcriptome.

In this study, we identified the genome sequence of the novel virus Pistacia-associated flexivirus 1 (PAFV1), a putative member of the mycovirus family Gammaflexiviridae (the order Tymovirales), via analysis of a transcriptome dataset for the mastic tree (Pistacia lentiscus, the family Anacardiaceae). PAFV1 was predicted to have three open reading frames (ORFs): ORF1, encoding a replicase (REP) with RNA-dependent RNA polymerase activity; ORF2, a movement protein (MP); and ORF3, a hypothetical protein. The PAFV1 REP sequence showed high similarity to those of three known members of the family Gammaflexiviridae i.e., Entoleuca gammaflexivirus 1 (EnFV1), Entoleuca gammaflexivirus 2 (EnFV2), and Botrytis virus F (BVF). A genome contig of the fungus Monosporascus cannonballus also contained a sequence of an endogenous virus similar to that of PAFV1. Sequence comparison and phylogenetic analysis indicated that PAFV1, EnFV1, and the endogenous virus of M. cannonballus formed a distinct subgroup (apart from EnFV2 and BVF), and may be the founding members of a novel genus in the family Gammaflexiviridae. Notably, MP sequences of PAFV1/EnFV1 showed similarity to the MP sequences of the mycovirus group called tobamo-like mycoviruses (an unassigned taxon), implying that genomic recombination occurred between members of the family Gammaflexiviridae and tobamo-like mycoviruses. Since PAFV1 is phylogenetically related to mycoviruses, PAFV1 may also be a mycovirus that infected a fungus associated with the mastic tree sample, which is evidenced by the presence of fungal ribosomal RNA sequences in the mastic tree transcriptome. Thus, the PAFV1 genome sequence may be useful in elucidating the genome evolution of Gammaflexiviridae and tobamo-like mycoviruses. Keywords: Pistacia-associated flexivirus 1; Gammaflexiviridae; mycovirus, mastic tree.

A new emaravirus discovered in Pistacia from Turkey.

High throughput sequencing was performed on total pooled RNA from six Turkish trees of Pistacia showing different viral symptoms. The analysis produced some contigs showing similarity with RNAs of emaraviruses. Seven distinct negative-sense, single-stranded RNAs were identified as belonging to a new putative virus infecting pistachio. The amino acid sequence identity compared to homologs in the genus Emaravirus ranged from 71% for the replicase gene on RNA1, to 36% for the putative RNA7 gene product. All the RNA molecules were verified in a pistachio plant by RT-PCR and conventional sequencing. Although the analysed plants showed a range of symptoms, it was not possible to univocally associate the virus with a peculiar one. The possible virus transmission by mite vector needs to be demonstrated by a survey, to observe spread and potential effect on yield in the growing areas of the crop.

Discovery of Viruses and Virus-Like Pathogens in Pistachio using High-Throughput Sequencing.

Pistachio (Pistacia vera L.) trees from the National Clonal Germplasm Repository (NCGR) and orchards in California were surveyed for viruses and virus-like agents by high-throughput sequencing (HTS). Analyses of sequence information from 60 trees identified a novel virus, provisionally named "Pistachio ampelovirus A" (PAVA), in the NCGR that showed low amino acid sequence identity (approximately 42%) compared with members of the genus Ampelovirus (family Closteroviridae). A putative viroid, provisionally named "Citrus bark cracking viroid-pistachio" (CBCVd-pis), was also found in the NCGR and showed approximately 87% similarity to Citrus bark cracking viroid (CBCVd, genus Cocadviroid, family Pospiviroidae). Both PAVA and CBCVd-pis were graft transmissible to healthy UCB-1 hybrid rootstock seedlings (P. atlantica × P. integerrima). A field survey of 123 trees from commercial orchards found no incidence of PAVA but five (4%) samples were infected with CBCVd-pis. Of 675 NCGR trees, 16 (2.3%) were positive for PAVA and 172 (25.4%) were positive for CBCVd-pis by reverse-transcription polymerase chain reaction. Additionally, several contigs across multiple samples exhibited significant sequence similarity to a number of other plant virus species in different families. These findings require further study and confirmation. This study establishes the occurrence of viral and viroid populations infecting pistachio trees.

Pistachio (Pistacia vera L.) is a new natural host of Hop stunt viroid.

Besides hop, Hop stunt viroid (HpSVd) infects many woody species including grapevine, citrus, peach, plum, apricot, almond, pomegranate, mulberry and jujube. Here, we report the first detection of HpSVd in pistachio (Pistacia vera L.). Samples corresponding to 16 pistachio cultivars were obtained from a nearby almond collection. From these samples, low molecular weight RNAs were extracted for double polyacrylamide gel electrophoresis, northern-blot analysis and reverse transcription polymerase chain reaction assays. HpSVd was detected in 4 of the 16 pistachio cultivars in the first year and in 6 in the second, being also detected in the almond collection. Examination of the nucleotide sequences of pistachio and almond isolates revealed 13 new sequence variants. Sequences from pistachio shared 92-96 % similarity with the first reported HpSVd sequence (GenBank X00009), and multiple alignment and phylogenetic analyses showed that one pistachio isolate (HpSVdPis67Jabari) clustered with the plum group, whereas all the others clustered with the hop, and the recombinants plum-citrus and plum-Hop/cit3 groups. By identifying pistachio as a new natural host, we confirm that HpSVd is an ubiquitous and genetically variable viroid that infects many different fruit trees cultivated worldwide.