Intergeneric recombination between a new, spinach-infecting curtovirus and a new geminivirus belonging to the genus Becurtovirus: first New World exemplar.

A novel curtovirus, spinach severe curly top virus (SSCTV), was associated with symptomatic spinach plants collected from a commercial field in south-central Arizona during 2009. In addition, a second viral molecule of about 2.9 kb from the same spinach plants was amplified, cloned and sequenced. The latter isolate, herein named spinach curly top Arizona virus (SCTAV), was found to share 77 % pairwise sequence identity with beet curly top Iran virus (BCTIV), a leafhopper-transmitted geminivirus that has been assigned to the new genus Becurtovirus. The SCTAV genome encodes three viral-sense genes, V1, V2, and V3, and two complementary-sense genes, C1 and C2. There was no evidence for the presence of either a C3 or C4 ORF in the genome sequence. The genome organization of SCTAV is not like that of New World curtoviruses but instead is similar to that of BCTIV, which, to date, is only known to be present in Iran. Consistent with this observation, SCTAV and BCTIV both contain the unusual nonanucleotide TAAGATT/CC and a replication-associated protein, Rep (or C1), that is more closely related to the mastrevirus Rep than to those of curtoviruses reported to date. Both SSCTV and SCTAV were found to have a recombinant genome containing sequences (AY548948) derived from ancestral SCTV sequences in the virion-sense portions of the genome. Agroinoculation of Nicotiana benthamiana (Domin) plants with the cloned genome of SCTAV resulted in infection of 95 % of the plants and the development of severe curling symptoms, whereas only 20 % of the SSCTV-inoculated plants were infected, developing only mild curling symptoms. When plants were co-inoculated with both viruses, the frequency of infection remained higher for SCTAV than for SSCTV (80 % vs. 20 %), indicating no evidence of synergistic effects between the two viruses with respect to efficiency of infection.

Comparative uptake of enteric viruses into spinach and green onions.

Root uptake of enteric pathogens and subsequent internalization has been a produce safety concern and is being investigated as a potential route of pre-harvest contamination. The objective of this study was to determine the ability of hepatitis A virus (HAV) and the human norovirus surrogate, murine norovirus (MNV), to internalize in spinach and green onions through root uptake in both soil and hydroponic systems. HAV or MNV was inoculated into soil matrices or into two hydroponic systems, floating and nutrient film technique systems. Viruses present within spinach and green onions were detected by RT-qPCR or infectivity assays after inactivating externally present viruses with Virkon(®). HAV and MNV were not detected in green onion plants grown up to 20 days and HAV was detected in only 1 of 64 spinach plants grown in contaminated soil substrate systems up to 20 days. Compared to soil systems, a drastic difference in virus internalization was observed in hydroponic systems; HAV or pressure-treated HAV and MNV were internalized up to 4 log RT-qPCR units and internalized MNV was shown to remain infectious. Understanding the interactions of human enteric viruses on produce can aid in the elucidation of the mechanisms of attachment and internalization, and aid in understanding risks associated with contamination events.

Survival and transfer of murine norovirus 1, a surrogate for human noroviruses, during the production process of deep-frozen onions and spinach.

The reduction of murine norovirus 1 (MNV-1) on onions and spinach by washing was investigated as was the risk of contamination during the washing procedure. To decontaminate wash water, the industrial sanitizer peracetic acid (PAA) was added to the water, and the survival of MNV-1 was determined. In contrast to onions, spinach undergoes a heat treatment before freezing. Therefore, the resistance of MNV-1 to blanching of spinach was examined. MNV-1 genomic copies were detected with a real-time reverse transcription PCR assay in PAA-treated water and blanched spinach, and PFUs (representing infectious MNV-1 units) were determined with a plaque assay. A < or = 1-log reduction in MNV-1 PFUs was achieved by washing onion bulbs and spinach leaves. More than 3 log PFU of MNV-1 was transmitted to onion bulbs and spinach leaves when these vegetables were washed in water containing approximately 5 log PFU/ml. No decline of MNV-1 occurred in used industrial spinach wash water after 6 days at room temperature. A concentration of 20 ppm of PAA in demineralized water (pH 4.13) and in potable water (pH 7.70) resulted in reductions of 2.88 +/- 0.25 and 2.41 +/- 0.18 log PFU, respectively, after 5 min of exposure, but no decrease in number of genomic copies was observed. No reduction of MNV-1 PFUs was observed on frozen onions or spinach during storage for 6 months. Blanching spinach (80 degrees C for 1 min) resulted in at least 2.44-log reductions of infectious MNV-1, but many genomic copies were still present.

Transcriptional analysis of complementary sense genes in Spinach curly top virus and functional role of C2 in pathogenesis.

Spinach curly top virus (SCTV), the fifth characterized Curtovirus species belonging to the family Geminiviridae, is an agriculturally significant plant pathogen representing an emerging disease threat in the southern United States. The SCTV genome comprises a single DNA chromosome of approximately 3.0 kb, with the potential to code for seven proteins larger than 10 kDa but which relies extensively on the host for replication and transcription of its genome. In this study, we have identified viral and complementary sense transcripts in SCTV-infected plants, confirming a bidirectional transcription strategy for SCTV. The most abundant RNA maps to the virion sense (1.1-kb transcript) and is comparable in size and location to that observed in Beet curly top virus (BCTV). Two complementary sense transcripts (1.7 and 0.7 kb) were identified in SCTV-infected plants. The large, 1.7-kb transcript is comparable in size and position to that identified in BCTV and several begomoviruses and most likely encodes the C1 protein. Both complementary sense RNAs could potentially direct expression of C2 and C3 from polycistronic mRNAs. A mutation in the C2 gene of SCTV results in expression of a truncated protein of 38 amino acids that is capable of interacting with two cellular kinases, AKIN11 and ADK2, and the resulting mutant virus remains highly infectious. A second mutant virus can only express the first three amino acids of the C2 protein and is unable to interact with the same kinases. However, this mutant virus still remains infectious, although a reduction in infectivity and symptom severity was seen in both Arabidopsis and spinach. A possible relationship between the interaction of C2 with AKIN11 and ADK2 and disease severity is presented.