Nuclear Inclusion Body Research Articles

First report of Nerine yellow stripe virus infecting crinium lily (Crinium sp.) in Texas.

Crinum sp. (family Amaryllidaceae) is an ornamental flower bulb that is commonly called crinum lily, cape lily, cemetery plant, spider lily, and swamp lily. In April 2023, two plants of Crinum sp. var. Maiden's Blush with yellow stripe symptoms (Fig. S1) were submitted to the Texas Plant Virus Diagnostic Laboratory, Weslaco, TX for virus diagnosis. Due to the resemblance of the observed symptoms to those described for potyviruses infecting ornamental flower bulbs (Pearson et al. 2009), total RNA extracts were made from each sample using the SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, USA), according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from 2 µg total RNA per sample with Oligo(dT) primers using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, USA) as recommended by the manufacturer. A 2µL aliquot of each cDNA template was initially subjected to PCR using the generic primer pair CIFor/CIRev (Ha et al., 2008) that targets a fragment of the cylindrical inclusion (CI) body of potyviruses. The expected ~700 bp DNA band was amplified from both samples using the Taq DNA polymerase, dNTPack kit (Sigma-Aldrich). The amplicons were cloned and sequenced (three recombinant clones per sample) as described by Hernandez et al. (2021) and the BLASTX analyses of the consensus sequence (GenBank acc. no. OR137018) returned significant hits only to nerine yellow stripe virus (NeYSV; Potyvirus, Potyviridae) at 100% query coverage. To further confirm the results, another pair of universal primers (Jordan et al. 2011) was used to amplify the expected ∼1,600 bp product specific to the partial nuclear inclusion body (NIb), coat protein (CP) cistron, and 3' untranslated region of potyviruses from the same samples. The amplicons were similarly cloned, and a consensus sequence obtained (OR137019). In pairwise comparisons, the partial CI sequence of NeYSV from Texas (NeYSV-TX; OR137018) shared 83% nucleotide (nt)/93% amino acids (aa) identities with the corresponding sequences of NeYSV isolate 63 (MT396083) from the United Kingdom. The partial (649 nt) NIb sequences of NeYSV-TX (OR137019) and the complete CP (OR137019) of NeYSV-TX shared 77-94%/88-94% and 83-99%/89-98% nt/aa identities with the corresponding sequences of global NeYSV isolates that were retrieved from GenBank. Phylogenetic analysis revealed a closer relationship between NeYSV-TX and the isolates Stenomesson (EU042758) and DC (MG012805) from the Netherlands and USA, respectively based on the partial NIb and CP cistrons (Fig. S2), suggesting that NeYSV-TX may have been introduced from foreign and/or domestic sources. NeYSV has been documented previously from the United Kingdom, the Netherlands, Australia, New Zealand, and India; its first report from the United States was a decade ago from Amaryllis belladonna in California (Guaragna et al. 2013). To the best of our knowledge, this is the first report of NeYSV in Texas, thus expanding the geographical range of the virus in the USA. Anecdotal information from the sample submitter implicated infected crinum lily bulbs as the likely source of NeYSV introduction into the property, with subsequent vegetative propagation of plants resulting in 100% incidence of symptomatic lilies (n>100) over time. Thus, the results underscore the importance of ensuring that only virus-free vegetative plant materials are distributed and propagated by florists to curtail virus spread.

Routine immunohistochemistry study for polyomavirus BK nephropathy in transplanted kidney biopsies, is it recommended?

BackgroundEarly diagnosis and treatment of Polyomavirus BK Nephropathy (PVBKN) is a challenging issue in the management of patients with kidney transplantation. Currently, histopathologic diagnosis is the gold standard method for diagnosis of PVBKN. However, typical viral inclusions may not be found in early stages of the PVBKN and should, instead, be diagnosed using immunohistochemistry (IHC) study. There is no clear consensus about routine IHC tests in the pathologic evaluation of transplanted kidney biopsy samples.Material and methodsThe current study was conducted on transplanted kidney biopsy samples, since 2016 to 2019. The patients who have presented with new onset of allograft dysfunction, at least 2 weeks after transplantation surgery, were included in our study. All these biopsy samples were evaluated with routine renal biopsy stains as well as IHC for SV40 (Simvian Virus 40) antigen. The identification of typical nuclear virus inclusion body and any nuclear positive staining on IHC (≥1+ positive result) were considered as definite evidence of PVBKN. Sensitivity, specificity, Positive Predictive and Negative Predictive Values (PPV and NPV) of histopathologic assessment without IHC study were evaluated.ResultsAmong 275 included cases, 18 (6.5%) patients with PVBKN were diagnosed. In patients with PVBKN, typical viral inclusions were detected in 14 samples (77.7%), on primary histopathological examination. However, virus-infected cells were identified just after IHC study in 4 (22.2%) of patients. Sensitivity, Specifity, PPV and NPV of morphologic histopathological assay without IHC for detection of PVBKN was 77.7, 100, 100 and 98.4% respectively.ConclusionRoutine IHC study for SV40 in all transplanted kidney biopsy samples with new onset of allograft dysfunction, will enhance the diagnostic sensitivity of early stage disease detection.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body ‘b’ and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

First Report of Bean Yellow Mosaic Virus Infecting Nasturtium (Tropaeolum majus) in Hawaii

Garden nasturtium (Tropaeolum majus L.) is an herbaceous annual plant commonly used as a garden ornamental, culinary herb, or a medicinal plant. It has become naturalized in many locations and is currently considered an alien invader in New Zealand, Lord Howe Island, and Hawaii (U.S. Forest Service 2012). Bean yellow mosaic virus (BYMV) is an aphid-transmitted nonpersistent potyvirus with a wide host range (Parrella and Lanave 2009). In February 2018, we observed nasturtium plants exhibiting severe chlorotic mosaic symptoms at the Kula Botanical Gardens on Maui, HI (20°44′40.01″ N, 156°19′03.00″ W). We tested six samples that had severe yellow mosaic and leaf distortion symptoms and three leaves from nonsymptomatic plants using potyvirus-specific ELISA (Agdia, Elkhart, IN) and universal potyvirus-specific reverse transcription polymerase chain reaction (RT-PCR) with potyvirus nuclear inclusion body (NIb) primers NIb2F and NIb3R (Zheng et al. 2010). All of the symptomatic nasturtium samples tested positive for potyvirus infection in both tests, whereas three nonsymptomatic nasturtium leaves tested negative in both tests. To identify the specific potyvirus involved, amplicons (309 bp) of the NIb region that were generated by RT-PCR using universal potyvirus-specific primers were sequenced (accession MH422546). BLASTn analysis of the sequences showed that this virus shared 93% nucleotide identity with a BYMV isolate from Australia (HG970862), and BLASTx analysis showed it shared 93% amino acid identity with a polyprotein of BYMV isolate from Australia (CDO67686). Two additional sets of PCR primers were used to further characterize the isolate: BYMV NIb-specific primers BYMV-NIb-3 (5′-CCACTGTTATTCCCTTTAAAC-3′) and BYMV-NIb-5 (5′-AATAAGTTCTACAGTTTGCACC-3′), and BYMV coat protein (CP)-specific primers BYMV-CP-3 and BYMV-CP-5 (Wang et al. 2017). BLASTn analysis of the 257-bp NIb amplicon (accession MH422547) shared 94% identity to an Australian isolate of BYMV (HG970847), and the 595-bp CP amplicon (accession MH422548) shared 98% identity to another Australian isolate of BYMV (HG970860). BLASTx analysis of the 257-bp NIb amplicon shared 93% identity to an Australian isolate polyprotein of BYMV (AHQ95554), and the 595-bp CP amplicon shared 98% identity to another Japan isolate polyprotein of BYMV (BAA93683). All six symptomatic samples also tested positive for BYMV by triple-antibody sandwich ELISA using a BYMV-specific antibody (DSMZ, Braunschweig, Germany). From March to June 2018, 24 additional nasturtium samples were collected from two different locations on Maui, and all tested positive for BYMV infection by RT-PCR and ELISA. All of the symptomatic nasturtium samples tested positive for BYMV infection in both tests, while three nonsymptomatic nasturtium leaves from other plants tested negative in both tests. To our knowledge, this is the first report of BYMV infecting nasturtium plants in Hawaii. Nasturtium is a widely grown herbaceous ornamental in Hawaii. It could be harboring BYMV in the off-season and moving it to cultivated legume plants. Therefore, further study is needed to determine whether legume aphids can transmit BYMV from nasturtium to healthy legume plants. In addition, further studies on the distribution of BYMV-infected nasturtium and identification of other BYMV-infected plants in the Hawaiian Islands are needed.

First Report of Apple of Peru (Nicandra physalodes) Infected with Pepper Mottle Virus in Hawaii

Apple of Peru (Nicandra physalodes [L.]) is a member of the Solanaceae family and one of the serious weed threats in crop fields. It is tolerant to commonly used herbicides and competes with cultivated crops (Watanabe et al. 2002). Additionally, apple of Peru and other solanaceous weeds are known as an alternative host of several viruses (Barbosa et al. 2009; Hobbs et al. 2000). Pepper mottle virus (PepMoV) in the genus Potyvirus (family Potyviridae) is a monopartite, positive-sensed, single-stranded RNA virus. PepMoV causes economic losses to peppers and tomatoes in Honduras, South Korea, and Hawaii (Kim et al. 2009; Melzer et al. 2012). In February 2018, we observed apple of Peru plants with severe virus-like symptoms: mosaic, mottling, and crinkling of leaves in a fallow area at the University of Hawaii in Manoa, HI. Six samples with severe symptoms and three asymptomatic samples were collected and tested for potyvirus infection using a potyvirus-specific ELISA (Agdia, Elkhart, IN) and universal potyvirus-specific reverse transcription polymerase chain reaction (RT-PCR) (Zheng et al. 2010). All of the symptomatic apple of Peru plants tested positive for potyviruses in both tests, whereas three asymptomatic plants tested negative. To identify the specific potyvirus involved, amplicons (328 bp) of the nuclear inclusion body (NIb) region of potyviruses that were generated by RT-PCR using universal potyvirus-specific primers were directly sequenced (GenBank accession MH476197). BLASTn analysis of the sequences showed that this virus isolate shared 95% nucleotide sequence identity with a PepMoV isolate from Florida (AF501591). Two additional primer pairs were used to further characterize the isolate: PepMoV coat protein (CP) gene primers 900/S (Melzer et al. 2012) and PepMoV NIb gene primers PMoVFP (5′-GTGATTTCAACAACCAGTTT-3′)/PMoVRP (5′-CCACTGTTATTGCCTCTGAA-3′) designed from the sequence generated by the universal potyvirus-specific primers (GenBank accession MH476197). The cDNA (1 µl) was subjected to PCR in a 20-µl reaction mixture containing 10 µl of GoTaq Master Mix (Promega) and 10 pmol of PMoVFP and PMoVRP primers. The thermal cycler program was as follows: 94°C for 5 min; followed by 35 cycles of amplification at 94°C for 30 s, 48°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min. BLASTn analysis of the 670-bp CP amplicon (GenBank accession MH499458) shared 98% nucleotide sequence identity to a Hawaii isolate of PepMoV (JQ429788) infecting tomato. BLASTn analysis of the 310-bp NIb amplicon (GenBank accession MH476198) shared 97% nucleotide sequence identity to a PepMoV isolate from Florida (AF501591). These samples also tested positive for PepMoV by triple-antibody sandwich (TAS) ELISA using PepMoV-specific antibodies (Agdia). In June 2018, nine new apple of Peru samples with severe symptoms and three asymptomatic samples were collected from the same area and tested again. All of the symptomatic apple of Peru plants tested positive for PepMoV in TAS-ELISA using PepMoV-specific antibodies, whereas three asymptomatic plants tested negative. This study is the first report of apple of Peru as a host of PepMoV in Hawaii. Apple of Peru is an invasive weed in tomato and pepper fields in Hawaii, and our results indicate eliminating solanaceous weeds is an important management step to mitigate potential losses by PepMoV in these commercial crops.

Surveys in the Gauteng, Limpopo and Mpumalanga provinces of South Africa reveal novel isolates of sweet potato viruses

Previous surveys carried out between 2001 and 2003 in the Limpopo, Mpumalanga, Gauteng, Western Cape, Eastern Cape, Kwazulu Natal and North West Provinces of South Africa reported Sweet potato feathery mottle virus and Sweet potato virus G as the most common viruses of sweet potato. The two potyviruses were detected by indexing onto the indicator plant Ipomoea setosa Kerr. and by nitrocellulose enzyme linked immunosorbent assays (NCM-ELISA). In this study, surveys (2012/2013 and 2013/2014) were conducted in three Provinces in South Africa, Gauteng, Limpopo and Mpumalanga. Results showed the frequent occurrence of the russet crack (RC) strain of Sweet potato feathery mottle virus, Sweet potato virus G, Sweet potato virus C, and begomoviruses Sweet potato leaf curl Sao Paulo virus, Sweet potato leaf curl virus and Sweet potato mosaic virus in the three Provinces surveyed. Potyvirus identities were confirmed by partial sequencing of the part of nuclear inclusion body (NIb) protein, coat protein (CP) gene and the 3′-non-translated region (3′ NTR) of Sweet potato feathery mottle virus and Sweet potato virus C, and the NIb and CP-encoding region of Sweet potato virus G. The RC strain of Sweet potato feathery mottle virus from this study shared nucleotide (nt) identity of between 97% to 99% with the RC strains from Australia, Peru and China, and 94% to 98% with isolates from New Zealand and United States of America. Sweet potato virus C isolates from this study shared 91% to 94% (VER-7_SPVC and REF-26_SPVC) and 86% to 91% (GBD-12_SPVC and MAM-15_SPVC) nt identity with the previously reported Sweet potato virus C isolates reported in South Africa and other parts of the world. Similarly, Sweet potato virus G samples from the current study shared 94% to 97% nt identity with the South African CTB3-3 and isolates from other countries, and genetic diversity was observed when samples of the current study shared the nt identity of 85% to 87% with the Kwazulu Natal isolates. The presence of begomoviruses was confirmed by observing for typical symptoms such as the upward curling and rolling of leaves on Ipomoea setosa. Rolling circle amplification (RCA) and next generation sequencing (NGS) of the full-length genome revealed novel begomoviruses isolates, which were identified as Sweet potato leaf curl Sao Paulo virus isolate Tanz: ELLP_ZA and Sweet potato leaf curl Sao Paulo virus isolate Tanz: NMP-ZA since the two shared 94% and 95% nt identity with Sweet potato leaf curl Sao Paulo virus isolate TZ-SNG7:12 of Tanzania, respectively. TMBLP-ZA appears to be a novel isolate of Sweet potato leaf curl virus since it shared 91% nt identity with Sweet potato leaf curl virus isolates from different geographic regions of the world. Therefore, we propose that it is classified as Sweet potato leaf curl virus isolate South Africa (ZA): TMBLP-ZA.

Frequency of nuclear mutant huntingtin inclusion formation in neurons and glia is cell-type-specific.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disorder that is caused by a CAG expansion in the Huntingtin (HTT) gene, leading to HTT inclusion formation in the brain. The mutant huntingtin protein (mHTT) is ubiquitously expressed and therefore nuclear inclusions could be present in all brain cells. The effects of nuclear inclusion formation have been mainly studied in neurons, while the effect on glia has been comparatively disregarded. Astrocytes, microglia, and oligodendrocytes are glial cells that are essential for normal brain function and are implicated in several neurological diseases. Here we examined the number of nuclear mHTT inclusions in both neurons and various types of glia in the two brain areas that are the most affected in HD, frontal cortex, and striatum. We compared nuclear mHTT inclusion body formation in three HD mouse models that express either full‐length HTT or an N‐terminal exon1 fragment of mHTT, and we observed nuclear inclusions in neurons, astrocytes, oligodendrocytes, and microglia. When studying the frequency of cells with nuclear inclusions in mice, we found that half of the population of neurons contained nuclear inclusions at the disease end stage, whereas the proportion of GFAP‐positive astrocytes and oligodendrocytes having a nuclear inclusion was much lower, while microglia hardly showed any nuclear inclusions. Nuclear inclusions were also present in neurons and all studied glial cell types in human patient material. This is the first report to compare nuclear mHTT inclusions in glia and neurons in different HD mouse models and HD patient brains. GLIA 2016;65:50–61

Association of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in India

HomePlant DiseaseVol. 99, No. 11Association of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in India PreviousNext DISEASE NOTES OPENOpen Access licenseAssociation of Nerine yellow stripe virus With Mosaic Disease of Crinum asiaticum Ornamental Plant in IndiaS. Kumar, R. Raj, C. Kaur, and S. K. RajS. KumarSearch for more papers by this author, R. RajSearch for more papers by this author, C. KaurSearch for more papers by this author, and S. K. RajSearch for more papers by this authorAffiliationsAuthors and Affiliations S. Kumar R. Raj C. Kaur S. K. Raj , Plant Molecular Virology Laboratory, CPMB Division, CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, Uttar Pradesh, India. Published Online:26 Aug 2015https://doi.org/10.1094/PDIS-02-15-0211-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Crinum asiaticum L. (Amaryllidaceae), commonly known as spider lily, is widely grown as an ornamental plant in gardens of tropical and subtropical countries for its long, strap-like, green leaves and clustered white flowers. During a survey in December 2013, severe mosaic symptoms were observed on a number of C. asiaticum plants growing in gardens at Noida and New Delhi cities of India. The symptoms were similar to those described earlier from Brazil on a potyvirus-infected Crinum sp. (Barboza et al. 2006); therefore, potyvirus infection was suspected. Electron microscopy was performed with leaf dip preparations of six randomly collected symptomatic leaf samples which revealed the presence of flexuous, rod-shaped, ∼700 × 12-nm virus particles, suggesting association of a potyvirus. These samples were further subjected to RT-PCR using potyvirus degenerate primers (Gibbs et al. 2003). RT-PCR resulted in the expected size (∼1.5 kb) amplicon in all symptomatic samples. The amplicons were cloned and sequenced, and sequence data were deposited as GenBank Accession Nos. KJ886933 (Nod-1), KM066970 (Nod-2), KM066968 (Nod-3), KJ886934 (Del-1), KM066971 (Del-2), and KM066969 (Del-3) isolates. Local alignments of the sequences of these isolates in NCBI revealed the presence of partial nuclear inclusion body (NIb), complete coat protein (CP) genes, and the 3′ UTR untranslated region (UTR) of a potyvirus. The isolates under study shared 98 to 99% sequence identity together and showed 78 to 99% identities with Nerine yellow stripe virus (NeYSV) isolates (FJ618537, DQ407932, EF362621, EF362622, EU042758, and JX865782) reported from New Zealand, The Netherlands, and USA. During phylogenetic analysis by MEGA (V.5.1) (Tamura et al. 2013), the isolates under study clustered together and showed close relationships with NeYSV isolates, and the potyvirus associated with mosaic disease of C. asiaticum was identified as NeYSV. The six and other nine symptomatic leaf samples of C. asiaticum were also tested by nucleic acid hybridization assay using the probe prepared from a clone of NeYSV (KJ886933), which gave strong signals of hybridization confirming presence of NeYSV in them. The probe may be used for the indexing of leaf samples collected from various locations and to understand the geographical distribution of NeYSV. A literature survey revealed the only report of association of an unidentified potyvirus with the mosaic disease of Crinum sp. from Brazil (Barboza et al. 2006) based on RT-PCR amplification of 2.0 kb product by potyvirus genus-specific primers. To best of our knowledge, this is the first report of natural occurrence of NeYSV on C. asiaticum in India. Because C. asiaticum is being propagated through bulbs, the dissemination of virus from nursery to nursery and from place to place is a major concern and now warrants the indexing of mother culture of C. asiaticum for NeYSV.

Molecular identification and expression of papaya ringspot virus gene in tobacco (Nicotiana benthamiana Domin) plants

Papaya ringspot virus (PRSV) belongs to Potyviridae family, genus Potyvirus and mainly transmitted by aphids (genera Macrosiphum and Myzus). Infected plants show mottling, blister-like patches and distortion of leaves as well as causes ring spot disease in papaya (Carica papaya L.) fruits. The HC-Pro (helper component proteinase), CI (cylindrical inclusion), NIb (nuclear inclusion body b) and Cp (coat protein) gene are involved in many other essential processes during the virus infection cycle as well as for transmission of potyvirus. Novel degenerate primers have been designed for amplification these genes. Phylogenetic analysis showed that the HC-Pro had the closest relationship with PRSV P isolate MP, CI with PRSV P isolate DEL and partial Nib plus CP with PRSV P isolate DEL as well as PRSV P isolate USA. Eluted PCR product of amplified gene was checked in 1 % agarose gel and cloned in binary vector with reporter gene (GUS). Agroinfiltration and GUS histochemical staining was performed for detecting the movement of a particular gene. The high sensitivity of GUS assays permitted temporal analysis of early expression of potyviral genes. Expression study proved that all genes are involved in long distance movement in host plants.

First Report of Catharanthus mosaic virus in Mandevilla in the United States.

Mandevilla (Apocynaceae) is an ornamental tropical vine popular for its bright and attractive flowers. During 2012 to 2013, 12 Mandevilla sp. samples from Minnesota and Florida nurseries were submitted for analysis at the University of Minnesota Plant Disease Clinic. Plants showed mosaic symptoms, leaf deformation, premature leaf senescence, and vine dieback. Filamentous virus particles with modal lengths 700 to 900 nm were observed by transmission electron microscopy (TEM) in partially purified preparations from symptomatic leaves. Partially purified virions were obtained using 30% sucrose cushion centrifuged at 109,000 gmax for 2 h at 10°C (5). No other virus particles were observed in these samples, nor were any observed in non-symptomatic samples. One sample was submitted as potted plant (Mandevilla 'Sunmandeho' Sun Parasol Giant White) and was kept under greenhouse conditions for subsequent analyses. Total RNA (Qiagen) was extracted from this sample, and Potyvirus was detected using the universal primers Poty S (5'-GGN AAY AAY AGY GGN CAR CC-3') and PV1 (5'-20(T)V-3') (1) by reverse transcription (RT)-PCR (3). The amplified product was the expected ~1.7-kb, corresponding to the partial nuclear inclusion body gene, the coat protein (CP) gene, and the 3' end untranslated region. The RT-PCR amplicon was cloned (NEB) and sequenced, and the 1,720-bp consensus sequence was deposited in GenBank (Accession No. KM243928). NCBI BLAST analysis at the nucleotide level revealed highest identity (83%) with an isolate of Catharanthus mosaic virus (CatMV) from Brazil (Accession No. DQ365928). Pairwise analysis of the predicted 256 amino acid CP revealed 91% identity with the CatMV Brazilian isolate (ABI94824) and 68% or less identity with other potyviruses. Two potyviruses are usually considered the same species if their CP amino acid sequences are greater than 80% identical (2). Serological analysis of the infected sample Mandevilla 'Sunmandeho' Sun Parasol Giant White using a CatMV specific antiserum (4) resulted in positive indirect ELISA reactions. CatMV has been previously reported in periwinkle (Catharanthus roseus) in Brazil (4). Based on the analyses by TEM, RT-PCR, nucleotide and amino acid sequence identities, and serological reactivity, we identify this virus as a U.S. Mandevilla isolate of CatMV. To our knowledge, this is the first report of Catharanthus mosaic virus both in the United States and in Mandevilla.

First Report of Nerine yellow stripe virus in Amaryllis in the United States.

Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers, and rhizomes) are increasingly important floriculture crops. Amaryllis is a small genus of flowering bulbs, with two species. The South African native, Amaryllis belladonna, also known as belladonna lily, Jersey lily, naked lady, Amarillo, or March lily, is one of numerous ornamental species with the common name "lily" due to their flower shape and growth habit. Amaryllis are popular for their 6- to 10-inch trumpet shaped colorful flowers that are borne on 1- to 2-foot stalks. In January, 2011, a home gardener in California observed mosaic symptoms on the leaves of A. belladonna growing in her garden. Leaf samples were sent to Agdia Inc. for testing. Samples tested positive for the presence of Potyvirus in a reverse transcription (RT)-PCR screen using universal potyvirus primers (2) yielding the expected ∼1,600-bp product corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene, and 3' end untranslated region (UTR). Electron microscopy of symptomatic leaves confirmed the presence of filamentous potyvirus-like particles. The RT-PCR amplicon was cloned and sequenced (2); the 1,616-bp consensus sequence was deposited in GenBank (Accession No. JX865782). NCBI BLAST analysis of the consensus sequence revealed highest identities with isolates of Nerine yellow stripe virus (NeYSV; family Potyviridae, genus Potyvirus). Pair-wise analyses of the 261 amino acid sequence of the predicted CP had 88% sequence identity with a Stenomesson isolate reported from the Netherlands (EU042758); 87% identity with Hymenocallis and Nerine isolates, both also from the Netherlands (EF362622 and EF362621, respectively); and, 86% with two New Zealand isolates infecting Amaryllis or Vallota (FJ618537 and DQ407932, respectively). The five Netherlands and New Zealand isolates are more closely related to each other than to the U.S. isolate as they share 93 to 98% CP identity. When using viral genome sequence relatedness as a criterion for defining potyvirus species, isolates with CP amino acid identity greater than 80% are considered the same species (1). The predicted coat protein gene of the California isolate was sub-cloned into the bacterial expression vector pET44 EK/LIC. Serological analysis of coat protein expressing clones in ELISA and Western Blot analysis using a potyvirus broad-spectrum reacting monoclonal antibody PTY-2 (3) and a NeYSV-specific rabbit antiserum (Applied Plant Research, Lisse, The Netherlands) resulted in positive reactions. NeYSV has previously been reported in the United Kingdom, the Netherlands, Australia, and New Zealand. Based on the results of electron microscopy, RT-PCR, nucleotide and amino acid identity, and serological reactivity, we identify this virus as a U.S. isolate of NeYSV, NeYSV-US. To our knowledge, this is the first report of Nerine yellow stripe virus in the United States. Development of antisera specific to this U.S. isolate is in progress.

Hippeastrum mosaic virus diagnosed in Hippeastrum and Eucharis in Brazil.

SUMMARY A potyvirus associated with foliar mosaic symptoms displayed by Hippeastrum (Hip-Br) and two Eucharis samples (Euch-B and Euch-C) from three different cities of Brazil was detected and identified by transmission tests, electron microscopy, reverse-transcription, RT-PCR and sequencing. Transmission electron microscopy revealed the presence of elongated, flexuous viral particles in the extracts of symptomatic leaves, while in thin sections of symptomatic leaves, cytoplasmic aggregates of type II lamellar inclusions (Edwardson’s classification) were observed. The experimental host range was restricted. RT-PCR using universal potyvirus primers amplified DNA products of 1,499 bp corresponding to the partial nuclear inclusion body (NIb) gene, full-length coat protein (CP) gene and 3’end untranslated region (UTR). Pairwise analyses from CP and NIb nucleotide sequences of Hip-Br, Euch-B and Euch-C viruses and other potyviruses revealed high identities with previously reported Alaska (GQ857550) and Rilona (EF203685) Hippeastrum mosaic virus (HiMV) isolates, Amazon lily mosaic virus (ALiMV, AY590143), and Amaryllis potyvirus (AmaPV, AY566239). The phylogeny calculated by a maximum likelihood method showed that Hip-Br, Euch-B and Euch-C formed a monophyletic group supported by 100% bootstrap with HiMV. This is the first report of HiMV in Hippeastrum sp. and Eucharis sp. from Brazil. Our data support the taxonomic position of HiMV and other potyviruses isolated from amaryllis and Amazon lily in a separated cluster of Amazon lily mosaic virus (AB158523).

First Report of Tobacco etch virus Infection in Coleus in the United States.

Virus-like disease symptoms consisting of foliar and veinal necrosis similar to those caused by Coleus vein necrosis virus (CVNV) (2) were observed in plants of coleus (Coleus blume Benth.) 'Rustic Orange' obtained from retail greenhouse outlets in Missouri and Minnesota. Flexuous, filamentous, 750 to 770 nm virus-like particles (vlps) were observed by transmission electron microscopy in negatively stained partially purified leaf tissue extracts from symptomatic 'Rustic Orange' leaf tissue. No other virus-like particles were observed and none were detected in extracts from asymptomatic leaves. These vlps were longer than those of CVNV (640 nm) (2) and were not detected by immunosorbent electron microscopy (ISEM) using antibodies to CVNV (2). Degenerate potyvirus primers PNIbF1 (5'GGBAAYAATAGTGGNCAACC3') and PCPR1 (5'GGGGAGGTGCCGTTCTCDATRCACCA3') (1) and total RNA extracted from 'Rustic Orange' leaf tissue with a Qiagen RNeasy Kit were used for reverse transcription-PCR with Ready-To-Go RT-PCR Beads (GE Healthcare). A 950-bp amplicon was obtained from total RNA from diseased but not from healthy leaf tissue. The nucleotide sequence of the amplicon (GenBank Accession No. GQ268818) had levels of identity to published Tobacco etch virus (TEV) sequences comprising portions of the nuclear inclusion body (NIb) and coat protein (CP) gene regions ranging from 89% (L38714) to 93% (M15239, M11458). The identity of the virus occurring in 'Rustic Orange' was further confirmed by ISEM. Virions were trapped and decorated by antibodies to TEV (ATCC PVAS 32). Systemically infected leaf tissue from Datura stramonium in which the coleus TEV isolate was propagated was used to mechanically inoculate Carborundum-dusted leaves of virus-free test plants of 'Rustic Orange' (Park Seed, Greenwood, SC). Inoculated plants developed foliar necrosis symptoms similar to those observed originally, and the presence of TEV was confirmed by ISEM and RT-PCR and nucleotide sequence analysis as described above. To our knowledge, this is the first report of a disease of coleus caused by TEV. Many of approximately 30 'Rustic Orange' plants in one nursery in Minnesota showed similar necrotic foliar symptoms and randomly selected plants tested positive for TEV by ISEM. This suggests that TEV infection in this variety may be spread by vegetative propagation from infected stock plants.

Plant microRNAs and their role in defense against viruses: a bioinformatics approach

BackgroundmicroRNAs (miRNAs) are non-coding short RNAs that regulate gene expression in eukaryotes by translational inhibition or cleavage of complementary mRNAs. In plants, miRNAs are known to target mostly transcription factors and are implicated in diverse aspects of plant growth and development. A role has been suggested for the miRNA pathway in antiviral defense in plants. In this work, a bioinformatics approach was taken to test whether plant miRNAs from six species could have antiviral activity by targeting the genomes of plant infecting viruses.ResultsAll plants showed a repertoire of miRNAs with potential for targeting viral genomes. The viruses were targeted by abundant and conserved miRNA families in regions coding for cylindrical inclusion proteins, capsid proteins, and nuclear inclusion body proteins. The parameters for our predicted miRNA:target pairings in the viral genomes were similar to those for validated targets in the plant genomes, indicating that our predicted pairings might behave in-vivo as natural miRNa-target pairings. Our screening was compared with negative controls comprising randomly generated miRNAs, animal miRNAs, and genomes of animal-infecting viruses. We found that plant miRNAs target plant viruses more efficiently than any other sequences, but also, miRNAs can either preferentially target plant-infecting viruses or target any virus without preference.ConclusionsOur results show a strong potential for antiviral activity of plant miRNAs and suggest that the miRNA pathway may be a support mechanism to the siRNA pathway in antiviral defense.

PVY-Resistant Transgenic Potato Plants Expressing an Anti-NIa Protein scFv Antibody

A synthetic gene encoding a single chain Fv fragment of an antibody directed against the nuclear inclusion a (NIa) protein of potato virus Y (PVY) was used to transform two commercial potato cultivars (Claustar and BF15). The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. Immunoblot analysis showed that most of the resulting transgenic plants accumulate high levels of the transgenic protein. Furthermore, a majority of the selected transgenic lines showed an efficient and complete protection against the challenge virus after mechanical inoculation with PVYO strain. Two transgenic lines showed an incomplete resistance with delayed appearance of symptoms accompanied by low virus titers, whereas one line developed symptoms during the first days after inoculation but recovered rapidly, leading to a low virus accumulation rate. These results confirm that expression of scFv antibody is able to inhibit a crucial step in the virus multiplication, such as polyprotein cleavage is a powerful strategy for engineered virus resistance. It can lead to a complete resistance that was not obtained previously by expression of scFv directed against the viral coat protein.

Ferritinopathy: diagnosis by muscle or nerve biopsy, with a note on other nuclear inclusion body diseases

Ferritinopathy (neuroferritinopathy) has recently been identified as an autosomal dominant, multisystem disease, mainly affecting the central nervous system. It is caused by mutations in exon 4 of the ferritin light chain gene on chromosome 19. Its fine structural hallmarks are granular nuclear inclusions in neurons, oligodendroglial and microglial cells with similar extracellular derivatives in the central nervous system, muscle, peripheral nerve, and skin. These pathognostic structures have previously been described in perivascular cells of muscle and nerve biopsy specimens in a case with an obviously identical disease, formerly described as 'granular nuclear inclusion body disease'. The nuclear inclusions, at the light microscopic level, are iron positive following histochemical iron reactions and immunoreactive for ferritin antibodies. At the electron microscopic level, in contrast to filamentous nuclear inclusions in 'neuronal intranuclear hyaline inclusion disease', dominant spinocerebellar atrophies and other trinucleotide repeat diseases, they are basically composed of granules measuring 5-15 nm. A moderate peak of iron detectable by energy dispersive microanalysis of the granular nuclear inclusions in ferritinopathy may also be significant. It is emphasized that ferritinopathy or 'granular nuclear inclusion body disease' can be diagnosed by a simple muscle or nerve biopsy without brain biopsy, autopsy, or molecular genetic testing of the considerable number of neurodegenerative diseases with possibly similar symptomatology.

The production of a genus-specific recombinant antibody (scFv) using a recombinant potyvirus protease

A single chain variable fragment antibody (scFv; anti-NIa scFv102) was selected from a synthetic human antibody library by using a NIa protease of Plum pox virus (PPV) as an antigen, which was expressed in bacteria. The NIa protease forms the nuclear inclusion body A and acts as the major protease in the cleavage of the viral polyprotein into functional proteins. The NIa protein was detected with anti-NIa scFv102 after expression in Escherichia coli cells as well as from PPV-infected Nicotiana benthamiana plants. Furthermore, the scFv102 has the ability to identify not only PPV from infected plants but also can detect other infections with members of the potyviruses. Nineteen different potyviruses were recognized by the scFv102 in various infected plants tested through dot blot assays. Therefore, the antibody scFv102 has the potential of becoming a general tool to detect potyvirus infections in different plant species.

Excessive apoptosis, increased phagocytosis, nuclear inclusion bodies and cylindrical confronting cisternae in bone marrow biopsies of myelodysplastic syndrome patients.

Transmission electron microscopic (TEM) studies have not been reported in bone marrow (BM) biopsies of patients with myelodysplastic syndromes (MDS) owing to failure to overcome the technical impediment of maintaining ultrastructural detail in decalcified tissue. Using a modified technique to physically separate pieces of bone from marrow tissue under a dissecting microscope, and embedding the material directly for TEM, ultrastructural studies were performed in 15 MDS patients and four normal BM biopsies. Biopsy tissue was also used to initiate long-term in vitro cultures and 12-week plates were sacrificed for TEM analysis. Features noted in freshly obtained decorticated tissue included an excessive apoptosis in both haematopoietic and stromal cells, ringed sideroblasts with iron-laden mitochondria and highly active, enormously increased phagocytosis. In addition, type IV nuclear inclusion body variants (NIB-v) and confronting cylindrical cisternae (CCC) were readily identified in up to 40% of stromal cells in vivo, providing an important footprint of a possible infectious agent in the pathology of MDS. Cultured stromal cells did not show excessive apoptosis and only 2-4% fibroblasts showed the presence of NIB-v or CCC, underscoring the artificial nature of ex vivo systems. We conclude that ultrastructure studies using decorticated tissue can be a powerful tool to investigate the biology and aetiology of a variety of haematopoietic disorders as it enables the direct examination of BM biopsies with their intimate stromal parenchymal cell associations preserved intact.

PENGGUNAAN PELACAK NONRADIOAKTIF (Digoxigenin-DNA Probe) UNTUK MENDETEKSI PEANUT STRIPE VIRUS

The use of nonradioactive probe (Digoxigenin-DNA) for detection of peanut stripe virus. The objective of this experiment was to develop the nonradioactive-labeled probe to detect peanut stripe virus (PStV) in peanut leaves and seeds. Digoxigenin labeled cDNA (dig-DNA probe) was synthesized from recombinant plasmid (pHS1.23) using polymerase chain reaction (PCR). The probe containing 1.195 bp (base pair) corresponding to 3' termini, included part of NIb (nuclear inclusion body) gene, coat protein gene, and 3' untranslated region of PStV genome was used to detect the existence of PStV in peanut leaves and seeds of infected peanut plants.

PENGKLONAN DAN PERUNUTAN NUKLEOTIDA GEN SELUBUNG PROTEIN DAN 3’UTR (untranslated region) PEANUT STRIPE VIRUS

Cloning and sequencing of coat protein gene and 3’UTR (untranslated region) of peanut stripe virus. The cDNA of 3' terminal of peanut stripe virus genomic RNA was cloned and sequenced. The cDNA was ligated with plasmid vector pGEM-T Easy and transformed to competent cells of Escherichia coli. The 3' terminal of PstV genomic RNA contained 1195 nucleotides (nts). The region included the nucleotide sequences of NIb (nuclear inclusion body) (129 nts), CP gene (coat protein gene) (861 nts), and 3'UTR (untranslated region) (205 nts). The nucleotide sequence of a CP gene contained one long uninterrupted open reading frame (ORF) without a start codon, which ended a UAG stop codon. The 287 amino acid residues of PStV coat protein were predicted from the CP gene. The amino acid was analyzed for the presence of consensus polyprotein cleavage site for maturation of potyvirus polyprotein. A putative cleavage site was found at position 43 (Q/S) following the Valine (V) residue at -4 position. This isolate of PstV can be expected to be aphid transmissible because the coat protein contained a DAG triplet at position 53-55.