Identification of grapevine Pinot gris virus in free-living Vitis spp. located in riparian areas adjacent to commercial vineyards

Plant Disease ◽  
2021 ◽  
Author(s):  
Alfredo Diaz-Lara ◽  
Gerald Dangl ◽  
Jydy Yang ◽  
Deborah Anne Golino ◽  
Maher Al Rwahnih
Keyword(s):  
Management Strategies ◽  
Rt Pcr ◽  
Free Living ◽  
Vitis Species ◽  
Reverse Transcription Pcr ◽  
Napa County ◽  
Grapevine Pinot Gris Virus ◽  
First Time ◽  
Infected Material ◽  
Vitis Spp

Grapevine Pinot gris virus (GPGV) is a recently identified pathogen of grapevines in California. To advance our knowledge about the epidemiology of GPGV, we investigated if free-living Vitis spp. can represent a source of virus infection. In 2019 a field survey of GPGV infection was conducted in Napa County. During the inspection 60 free-living vines in riparian habitats near commercial vineyards with GPGV infection were sampled. Samples were tested by real-time reverse transcription PCR (RT-PCR), identifying 23 free-living Vitis spp. positive for GPGV. Later, GPGV infection was confirmed in these plants via end-point RT-PCR and Sanger sequencing. Based on sequence analysis, detected GPGV isolates are more related to the asymptomatic variant of the virus. Vitis species ancestry was determined by DNA fingerprinting. GPGV-infected material included V. californica, V. californica × V. vinifera hybrids and hybrid rootstock cultivars. Here, GPGV is reported for the first time in free-living Vitis spp. The results of this study will support the development of management strategies for GPGV in California and beyond.

scholarly journals HTS-Based Monitoring of the Efficiency of Somatic Embryogenesis and Meristem Cultures Used for Virus Elimination in Grapevine

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1782
Author(s):  
Mihaly Turcsan ◽  
Emese Demian ◽  
Tunde Varga ◽  
Nikoletta Jaksa-Czotter ◽  
Erno Szegedi ◽  
...  
Keyword(s):  
Somatic Embryogenesis ◽  
High Throughput Sequencing ◽  
Vitis Vinifera L ◽  
Meristem Culture ◽  
Rt Pcr ◽  
Virus Elimination ◽  
Mother Plants ◽  
Virus Diagnostics ◽  
Grapevine Pinot Gris Virus ◽  
First Time

Meristem culture and somatic embryogenesis are effective tools for virus elimination of vegetatively propagated crops including grapevine (Vitis vinifera L.). While both have been shown to be useful to eliminate the main grapevine viruses, their efficiency differs depending on the virus and grapevine variety. In our work, we investigated the efficiency of these two virus elimination methods using small RNA high-throughput sequencing (HTS) and RT-PCR as virus diagnostics. Field grown mother plants of four clones representing three cultivars, infected with different viruses and viroids, were selected for elimination via somatic embryogenesis (SE) and meristem culture (ME). Our results show for the first time that using SE, elimination in mother plants was effective for all viruses, i.e., grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus 1 (GSyV-1), Grapevine virus T (GVT) and grapevine Pinot gris virus (GPGV). This study also confirms previous studies showing that SE is a possible strategy for the elimination of GFkV, GRSPaV, HSVd, and GYSVd-1. Our results demonstrate that the efficacy of virus elimination via SE is relatively high while the purging of viroids is lower. Our work provides evidence that the efficiency of SE is comparable to that of the technically difficult ME technique, and that SE will offer a more effective strategy for the production of virus-free grapevine in the future.

scholarly journals Detection of Tilapia Lake Virus in Clinical Samples by Culturing and Nested Reverse Transcription-PCR

2016 ◽  
Vol 55 (3) ◽  
pp. 759-767 ◽  
Author(s):  
Japhette Esther Kembou Tsofack ◽  
Rachel Zamostiano ◽  
Salsabeel Watted ◽  
Asaf Berkowitz ◽  
Ezra Rosenbluth ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Samples ◽  
Fish Cell ◽  
Rt Pcr ◽  
Fish Cell Lines ◽  
Pcr Assay ◽  
Farmed Fish ◽  
Reverse Transcription Pcr ◽  
Aquaculture Industry ◽  
First Time

ABSTRACT Tilapia are an important group of farmed fish that serve as a significant protein source worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here, we provide the conditions for efficient isolation, culturing, and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested reverse transcription-PCR (RT-PCR) assay allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time to our knowledge, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry with important tools for the detection and containment of this pathogen.

Detection of Nucleic Acids in Cells or Tissue Sections Using In situ Polymerase Chain Reaction (PCR)

1996 ◽  
Vol 54 ◽  
pp. 16-17
Author(s):  
J. R. Hully ◽  
K. R. Luehrsen ◽  
K. Aoyagi ◽  
C. Shoemaker ◽  
R. Abramson
Keyword(s):  
Nucleic Acids ◽  
Viral Infections ◽  
Anatomical Location ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Cellular Source ◽  
Gene Transcripts ◽  
Initial Description ◽  
In Cells

The development of PCR technology has greatly accelerated medical research at the genetic and molecular levels. Until recently, the inherent sensitivity of this technique has been limited to isolated preparations of nucleic acids which lack or at best have limited morphological information. With the obvious exception of cell lines, traditional PCR or reverse transcription-PCR (RT-PCR) cannot identify the cellular source of the amplified product. In contrast, in situ hybridization (ISH) by definition, defines the anatomical location of a gene and/or it’s product. However, this technique lacks the sensitivity of PCR and cannot routinely detect less than 10 to 20 copies per cell. Consequently, the localization of rare transcripts, latent viral infections, foreign or altered genes cannot be identified by this technique. In situ PCR or in situ RT-PCR is a combination of the two techniques, exploiting the sensitivity of PCR and the anatomical definition provided by ISH. Since it’s initial description considerable advances have been made in the application of in situ PCR, improvements in protocols, and the development of hardware dedicated to in situ PCR using conventional microscope slides. Our understanding of the importance of viral latency or viral burden in regards to HIV, HPV, and KSHV infections has benefited from this technique, enabling detection of single viral copies in cells or tissue otherwise thought to be normal. Clearly, this technique will be useful tool in pathobiology especially carcinogenesis, gene therapy and manipulations, the study of rare gene transcripts, and forensics.

scholarly journals Reassortment of Genome Segments Creates Stable Lineages Among Strains of Orchid Fleck Virus Infecting Citrus in Mexico

2020 ◽  
Vol 110 (1) ◽  
pp. 106-120 ◽  
Author(s):  
Avijit Roy ◽  
Andrew L. Stone ◽  
Gabriel Otero-Colina ◽  
Gang Wei ◽  
Ronald H. Brlansky ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sensu Stricto ◽  
Genome Segment ◽  
Rt Pcr ◽  
Sequence Comparisons ◽  
Orchid Fleck Virus ◽  
Reverse Transcription Pcr ◽  
Sequencing Technologies ◽  
Negative Sense

The genus Dichorhavirus contains viruses with bipartite, negative-sense, single-stranded RNA genomes that are transmitted by flat mites to hosts that include orchids, coffee, the genus Clerodendrum, and citrus. A dichorhavirus infecting citrus in Mexico is classified as a citrus strain of orchid fleck virus (OFV-Cit). We previously used RNA sequencing technologies on OFV-Cit samples from Mexico to develop an OFV-Cit–specific reverse transcription PCR (RT-PCR) assay. During assay validation, OFV-Cit–specific RT-PCR failed to produce an amplicon from some samples with clear symptoms of OFV-Cit. Characterization of this virus revealed that dichorhavirus-like particles were found in the nucleus. High-throughput sequencing of small RNAs from these citrus plants revealed a novel citrus strain of OFV, OFV-Cit2. Sequence comparisons with known orchid and citrus strains of OFV showed variation in the protein products encoded by genome segment 1 (RNA1). Strains of OFV clustered together based on host of origin, whether orchid or citrus, and were clearly separated from other dichorhaviruses described from infected citrus in Brazil. The variation in RNA1 between the original (now OFV-Cit1) and the new (OFV-Cit2) strain was not observed with genome segment 2 (RNA2), but instead, a common RNA2 molecule was shared among strains of OFV-Cit1 and -Cit2, a situation strikingly similar to OFV infecting orchids. We also collected mites at the affected groves, identified them as Brevipalpus californicus sensu stricto, and confirmed that they were infected by OFV-Cit1 or with both OFV-Cit1 and -Cit2. OFV-Cit1 and -Cit2 have coexisted at the same site in Toliman, Queretaro, Mexico since 2012. OFV strain-specific diagnostic tests were developed.

scholarly journals Phylogenomic Evidence of Reinfection and Persistence of SARS-CoV-2: First Report from Colombia

Vaccines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 282
Author(s):  
Juan David Ramírez ◽  
Marina Muñoz ◽  
Nathalia Ballesteros ◽  
Luz H. Patiño ◽  
Sergio Castañeda ◽  
...  
Keyword(s):  
Viral Persistence ◽  
Transmission Dynamics ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Paired Samples ◽  
Infection Dynamics ◽  
Oxford Nanopore ◽  
Novel Variants ◽  
First Time ◽  
Oxford Nanopore Technologies

The continuing evolution of SARS-CoV-2 and the emergence of novel variants have raised concerns about possible reinfection events and potential changes in the coronavirus disease 2019 (COVID-19) transmission dynamics. Utilizing Oxford Nanopore technologies, we sequenced paired samples of three patients with positive RT-PCR results in a 1–2-month window period, and subsequent phylogenetics and genetic polymorphism analysis of these genomes was performed. Herein, we report, for the first time, genomic evidence of one case of reinfection in Colombia, exhibiting different SARS-CoV-2 lineage classifications between samples (B.1 and B.1.1.269). Furthermore, we report two cases of possible viral persistence, highlighting the importance of deepening our understanding on the evolutionary intra-host traits of this virus throughout different timeframes of disease progression. These results emphasize the relevance of genomic surveillance as a tool for understanding SARS-CoV-2 infection dynamics, and how this may translate effectively to future control and mitigations efforts, such as the national vaccination program.

scholarly journals First Report of Echinococcus ortleppi in Free-Living Wild Boar (Sus scrofa) from Portugal

2021 ◽  
Vol 9 (6) ◽  
pp. 1256
Author(s):  
Teresa Letra Mateus ◽  
Maria João Gargaté ◽  
Anabela Vilares ◽  
Idalina Ferreira ◽  
Manuela Rodrigues ◽  
...  
Keyword(s):  
Wild Boar ◽  
Internal Transcribed Spacer ◽  
Sus Scrofa ◽  
One Health ◽  
Control Strategies ◽  
Free Living ◽  
First Report ◽  
Wildlife Reservoirs ◽  
First Time ◽  
Echinococcus Granulosus Sensu Lato

Cystic echinococcosis (CE) is a zoonosis that is prevalent worldwide. It is considered endemic in Portugal but few studies have been performed on Echinococcus granulosus sensu lato and their hosts. In this study, CE cysts are reported for the first time in a free-living wild boar (Sus scrofa) in Portugal. The presence of the metacestodes in the liver of the wild boar was identified by morphological features, microscopic examination and molecular analysis. The sequencing of part of the DNA nuclear ribosomal internal transcribed spacer-1 (ITS-1) region revealed a G5 genotype that presently corresponds to Echinococcus ortleppi. This is the first report of E. ortleppi in Portugal and to the best of the authors’ knowledge, in Europe. These results suggest that wild boar may be a host of CE, namely, crossing the livestock–wildlife interface, which has important public health implications. Wildlife reservoirs must be taken into account as CE hosts and surveillance of game as well as health education for hunters should be implemented using a One Health approach, with implementation of feasible and tailor-made control strategies, namely, proper elimination of byproducts in the field.

scholarly journals Molecular epidemiology of Crimean-Congo haemorrhagic fever virus in India

2016 ◽  
Vol 144 (16) ◽  
pp. 3422-3425 ◽  
Author(s):  
P. SINGH ◽  
M. CHHABRA ◽  
P. SHARMA ◽  
R. JAISWAL ◽  
G. SINGH ◽  
...  
Keyword(s):  
Eastern Europe ◽  
N Gene ◽  
Western India ◽  
Haemorrhagic Fever ◽  
Rt Pcr ◽  
Study Region ◽  
Phylogenetic Relatedness ◽  
Cchf Virus ◽  
Crimean Congo Haemorrhagic Fever ◽  
First Time

SUMMARYCrimean-Congo haemorrhagic fever (CCHF) is an emerging zoonotic disease in India which is prevalent in neighbouring countries. CCHF virus (CCHFV) is a widespread tick-borne virus which is endemic in Africa, Asia, Eastern Europe and the Middle East. In the present study, samples of clinically suspected human cases from different areas of northern-western India were tested for the presence of CCHFV by RT–PCR through amplification of nucleocapsid (N) gene of CCHFV. Positive samples were sequenced to reveal the prevailing CCHFV genotype(s) and phylogenetic relatedness. A phylogenetic tree revealed the emergence of diverse strains in the study region showing maximum identity with the Pakistan, Afghanistan and Iran strains, which was different from earlier reported Indian strains. Our findings reveal for the first time the emergence of the Asia 1 group in India; while earlier reported CCHFV strains belong to the Asia 2 group.

scholarly journals Milk Protein Fragments Induce the Biosynthesis of Macedocin, the Lantibiotic Produced by Streptococcus macedonicus ACA-DC 198

2009 ◽  
Vol 76 (4) ◽  
pp. 1143-1151 ◽  
Author(s):  
Marina Georgalaki ◽  
Marina Papadelli ◽  
Elina Chassioti ◽  
Rania Anastasiou ◽  
Anastassios Aktypis ◽  
...  
Keyword(s):  
Milk Protein ◽  
Mass Spectrometric ◽  
Partial Purification ◽  
Protein Fragments ◽  
Reverse Transcription Pcr ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Tandem Mass Spectrometric ◽  
Protein Components ◽  
First Time

ABSTRACT The aim of the present work was to study the mode of the induction of the biosynthesis of macedocin, the lantibiotic produced by Streptococcus macedonicus ACA-DC 198. Macedocin was produced when the strain was grown in milk but not in MRS or M17 broth. No autoinduction mechanism was observed. Production did not depend on the presence of lactose or galactose in the culture medium or on a coculture of the producer strain with macedocin-sensitive or macedocin-resistant strains. Induction seemed to depend on the presence of one or more heat-stable protein components produced when S. macedonicus ACA-DC 198 was grown in milk. The partial purification of the induction factor was performed by a combination of chromatography methods, and its activity was confirmed by a reverse transcription-PCR approach (RT-PCR). Mass spectrometric (MS) and tandem mass spectrometric (MS/MS) analyses of an induction-active fraction showed the presence of several peptides of low molecular mass corresponding to fragments of αS1- and β-casein as well as β-lactoglobulin. The chemically synthesized αS1-casein fragment 37-55 (2,253.65 Da) was proven to be able to induce macedocin biosynthesis. This is the first time that milk protein degradation fragments are reported to exhibit a bacteriocin induction activity.

Arthropods in the nests of lesser spotted eagle (Aquila pomarina)

Biologia ◽  
2009 ◽  
Vol 64 (5) ◽  
Author(s):  
Ján Krištofík ◽  
Peter Mašán ◽  
Zbyšek Šustek ◽  
Dušan Karaska
Keyword(s):  
Trophic Group ◽  
Trophic Groups ◽  
Significant Component ◽  
Free Living ◽  
Parasitic Species ◽  
Number Of Species ◽  
Aquila Pomarina ◽  
Investigation Period ◽  
First Time

AbstractIn 2001–2007, altogether 57 nests of lesser spotted eagle were collected in the Orava region in northwestern Slovakia and four groups of arthropods were extracted from them. Richest in number of species and individuals were mites (23 species, 17,500 ind.), followed by beetles (12 species, 725 ind.), whereas pseudoscorpions were represented only by Pselaphochernes scorpioides (39 ind.) and fleas by Ceratophyllus garei (3 ind.). Unlike nests of other birds, free-living mites predominated in the nests fauna (83% of individuals), followed by nidicolous species with more or less free relationship to the nests, while parasitic species represented only a negligible part of the fauna. For the first time we observed phoresy of Nenteria pandioni, a specific and abundant mite in the eagles’ nests, on the nidicolous staphylinid Haploglossa puncticollis. The beetle fauna in the nests was much poorer than in nests of other birds. The predatory H. puncticollis was dominant in the nests (83%) and occurred continuously during the whole investigation period. Other beetles, even the widely distributed nidicols such as the histerid Gnathoncus buyssoni, were found rarely in nests. Predators were also the only abundant trophic group of beetles in the nests, while other trophic groups of beetles abundantly co-occur in nests of majority of other birds. The occurrence of all beetles was very unevenly distributed during the investigation period, but was positively correlated with occurrence of mites. The relatively low number of species and individuals of mites and beetles in the lesser spotted eagle nests resulted from their position on tree tops, at a height of 20–30 m and their quick drying out by sun and wind. It was also indicated by an enormously low number of species and individuals of mycetophagous beetles, which represent a significant component of the fauna in nests of other birds.

Comet and cup genes in Drosophila spermatogenesis: the first demonstration of post-meiotic transcription

2008 ◽  
Vol 36 (3) ◽  
pp. 540-542 ◽  
Author(s):  
Carine Barreau ◽  
Elizabeth Benson ◽  
Helen White-Cooper
Keyword(s):  
In Situ Hybridization ◽  
Expression Pattern ◽  
Reverse Transcription ◽  
Protein Product ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Cyst ◽  
Reverse Transcription Pcr

Post-meiotic transcription is widespread in mammalian spermatogenesis, but is generally believed to be absent from Drosophila spermatogenesis. Genes required during meiosis, in early spermatids or later in spermiogenesis are typically transcribed in primary spermatocytes in Drosophila. Their mRNAs are then stored in the cytoplasm until the protein product is needed. Recently, using in situ hybridization, we identified 17 Drosophila genes, collectively named ‘comets’ and ‘cups’, whose mRNAs are most abundant in, and localize to the distal ends of, elongating spermatids. Using a single-cyst quantitative RT–PCR (reverse transcription–PCR) assay, we confirmed this unusual expression pattern and conclusively demonstrate the existence of post-meiotic transcription in Drosophila spermatids. We found that transcription of comets and cups occurs just before protamines can be detected in spermatid nuclei.

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