Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified. One of those species, GLRaV-7, was first reported from a symptomless white-fruited wine grape cultivar from Albania. Since its discovery, GLRaV-7 has been reported from 14 countries. Although serological assays have been developed to detect GLRaV-7, commercially available antibodies produce high background signals making them unsuitable for regulatory testing. Furthermore, while molecular detection assays have been shown to be more sensitive when compared to the serological assays, published molecular assays, except the one Reverse Transcription-quantitaive Polymerase Chain Reaction (RT-qPCR) assay based on heat shock protein 70 homologue (HSP70h) gene, have been reported to be inadequate in detecting all reported isolates of GLRaV-7. Availability of multiple assays provides flexibility to diagnostic laboratories in cases where the chosen assay fails to detect a strain or an isolate of a pathogen due to variation in its targeted region or where additional confirmation of the results is required. In this study, we developed a sensitive and specific RT-qPCR assay, based on a region of p61 gene of GLRaV-7, which detected all available isolates.

Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B.

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.

Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus.

We report the complete nucleotide sequences of lettuce infectious yellows virus (LIYV) RNAs 1 and 2. LIYV RNA 1 is 8118 nucleotides and includes three open reading frames (ORFs). Computer-assisted analysis of LIYV RNA 1 ORFs identified domains for a papain-like protease, methyltransferase (MTR), RNA helicase (HEL), and RNA-dependent RNA polymerase (RdRp). We suggest that the RdRp domain is expressed independently of the other replication-associated domains via a + 1 ribosomal frameshift. Amino acid sequences of the MTR, HEL, and RdRp show highly significant similarity to the homologous sequences from other closteroviruses and lower similarity to the respective proteins of tobamoviruses, tobraviruses, hordeiviruses, bromoviruses, and furoviruses. LIYV RNA 2 is 7193 nucleotides and includes six ORFs. These ORFs include a gene array that is characteristic of the closteroviruses: ORFs encoding a small membrane protein, a homologue of the HSP70 family of chaperone proteins, a protein whose function is unknown, the coat protein, and a diverged duplicate of the coat protein. LIYV is distinguished from the monopartite closteroviruses in the following ways: its genome consists of two RNAs, the positions of the coat protein gene and its diverged duplicate are reversed, and LIYV includes ORFs that are unrelated to ORFs found in other closteroviruses.

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2. To assess the relationships between LIYV RNAs 1 and 2, cDNAs corresponding to the virion RNAs were cloned. Northern blot hybridization analysis showed no detectable sequence homology between these RNAs. A partial amino acid sequence obtained from purified LIYV coat protein was found to align in the most upstream of four complete open reading frames (ORFs) identified in a LIYV RNA 2 cDNA clone. The identity of this ORF was confirmed as the LIYV coat protein gene by immunological analysis of the gene product expressed in vitro and in Escherichia coli. Computer analysis of the LIYV coat protein amino acid sequence indicated that it belongs to a large family of proteins forming filamentous capsids of RNA plant viruses. The LIYV coat protein appears to be most closely related to the coat proteins of two closteroviruses, beet yellows virus and citrus tristeza virus.

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA). In vitro translation of transcripts derived from cDNA clones representing regions of each ORF was used to identify the respective proteins. A ca. 22,000 Mr protein was translated from the 591-nt ORF transcript. This protein comigrated in SDS-PAGE with a protein produced by in vitro translation of MStV RNA and is referred to as the NS3 protein. The protein from the 948-nt ORF transcript was specifically immunoprecipitated by antiserum to the MStV nucleocapsid protein (N protein). RNA hybridization analyses identified two subgenomic RNAs in total RNA extracts from MStV-infected Zea mays plants. These RNAs are ca. 650 and 1350 nt in size, are of opposite polarities, and correspond to regions of RNA3 containing the two ORFs. These data suggest that MStV RNA3 has an ambisense coding strategy similar to that found for the S RNA of the vertebrate-infecting phleboviruses, uukuviruses, and arenaviruses, as for tomato spotted wilt virus.

Identification and sequence analysis of the maize stripe virus major noncapsid protein gene.

The maize stripe virus (MStV) major noncapsid protein (NCP) gene was characterized, and the location of the NCP gene was identified among the 5-RNA, 18-kb genome. A 12-amino-acid sequence of the NCP was compared with nucleotide sequence data for MStV RNAs 3 and 4 and was found to align perfectly within a 528-nucleotide open reading frame (ORF) of RNA 4. The amino acid composition of purified NCP was almost identical to that deduced from the putative coding region. The deduced NCP molecular weight was 19,815, very similar to that determined by SDS-PAGE analysis of the purified NCP. In vitro transcription and translation analysis of the cDNA representing this region showed unequivocally that this region encoded the NCP. Primer extension analysis using a synthetic oligonucleotide complementary to a sequence near the 5' end of the coding region revealed that the NCP ORF is located 61 nucleotides from the 5' end of RNA 4.

Complementary DNA cloning and hybridization analysis of maize stripe virus RNAs.

Cloned cDNAs were used to assess the relationships of the RNAs isolated from maize stripe virus (MStV) virion-like nucleoproteins. Northern blot hybridization analysis showed no detectable sequence homology between the five different sized RNAs; however, cDNAs which hybridized with a specific ssRNA also hybridized with a specific dsRNA. Hybridization analysis of denatured MStV RNAs using ssRNA riboprobes of opposite polarities showed that for each of the five MStV RNAs, opposite, complementary polarity RNAs were present. However, unequal amounts of complementary polarities for each size RNA were detected, and the polarity of the RNA in excess was primarily detectable as ssRNA.