scholarly journals Diversity of Ampeloviruses in Mealybug and Soft Scale Vectors and in Grapevine Hosts from Leafroll-Affected Vineyards

2009 ◽  
Vol 99 (10) ◽  
pp. 1177-1184 ◽  
Author(s):  
M. Fuchs ◽  
P. Marsella-Herrick ◽  
G. M. Loeb ◽  
T. E. Martinson ◽  
H. C. Hoch
Keyword(s):  
New York ◽  
Heat Shock Protein 70 ◽  
Host Plants ◽  
Protein Gene ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Pseudococcus Maritimus

The occurrence and diversity of Grapevine leafroll-associated virus 1 (GLRaV-1) and Grapevine leafroll-associated virus 3 (GLRaV-3) in the soft scales Parthenolecanium corni and Pulvinaria innumerabilis and in the mealybug Pseudococcus maritimus was determined in leafroll-affected vineyards in the Finger Lakes region of New York. Groups of 1 to 4 specimens were collected under loose grapevine bark and tested by reverse-transcription polymerase chain reaction (RT-PCR) for segments of the second diverged copy of the GLRaV-1 coat protein gene or GLRaV-3 heat-shock protein 70-homologue gene. Virus-specific RT-PCR products were amplified from immature insect vectors and adult mealybugs. Single viral amplicons were obtained mostly from immature vectors (35%, 30 of 85) and dual viral amplicons from immature (16%, 10 of 61) and adult (100%, 14 of 14) mealybugs, including individuals. These observations suggested a simultaneous uptake of GLRaV-1 and GLRaV-3 by individual mealybugs. Furthermore, a comparative nucleotide sequence analysis of viral amplicons from soft scales, mealybugs, and grapevines from which vectors were collected showed identical or highly similar haplotypes, indicating that uptake of GLRaV-1 and GLRaV-3 likely occurred by direct feeding of vectors on their host plants.

scholarly journals Use of Degenerate Primers for Partial Sequencing and RT-PCR-Based Assays of Grapevine Leafroll-Associated Viruses 4 and 5

1998 ◽  
Vol 88 (11) ◽  
pp. 1238-1243 ◽  
Author(s):  
Geoffrey Routh ◽  
Yun-Ping Zhang ◽  
Pasquale Saldarelli ◽  
Adib Rowhani
Keyword(s):  
Heat Shock Protein 70 ◽  
Plasmid Vector ◽  
Pcr Primers ◽  
Pcr Detection ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Double Stranded Rna ◽  
Pcr Products ◽  
Polymerase Chain

Double-stranded RNA (dsRNA) was purified from grapevines infected with grapevine leafroll-associated viruses 4 (GLRaV-4) and 5 (GLRaV-5), two putative closteroviruses. Reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on this dsRNA using degenerate oligonucleotides designed to amplify an approximately 550- to 650-nucleotide fragment from the heat shock protein 70 homolog (HSP70) of the known closteroviruses. RT-PCR products of the appropriate molecular weight were gel-isolated and cloned into the plasmid vector pGEM-T. Clones of RT-PCR products generated by using these primers on dsRNA isolated from a plant infected with GLRaV-4 were sequenced. This sequence was used to develop an immunocapture RT-PCR (IC-RT-PCR) detection protocol capable of detecting GLRaV-4. Similar clones were made from dsRNA isolated from a plant infected with GLRaV-5. These clones were also sequenced. The two sequences were compared, and RT-PCR primers were developed that were able to amplify cDNA from both. These experiments demonstrate that degenerate primers that amplify closterovirus HSP70 sequences can be used to successfully generate sequences useful for IC-RT-PCR detection of these viruses. These data also suggest that it is feasible to use HSP70 sequences to design PCR primers capable of more general PCR detection of multiple GLRaV serotypes. Lastly, the presence of closterovirus-like HSP70 sequences in these putative closteroviruses implies that they are indeed members of this taxonomic group.

scholarly journals Limited Genetic Variability Among American Isolates of Grapevine virus E from Vitis spp.

Plant Disease ◽  
2016 ◽  
Vol 100 (1) ◽  
pp. 159-163 ◽  
Author(s):  
J. Vargas-Asencio ◽  
M. Al Rwahnih ◽  
A. Rowhani ◽  
F. Celebi-Toprak ◽  
J. R. Thompson ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
New York ◽  
Protein Gene ◽  
The United States ◽  
Nucleic Acid Binding ◽  
Grapevine Virus ◽  
Rt Pcr ◽  
Viral Cdna ◽  
Polymerase Chain ◽  
Binding Protein Gene

A survey for the presence of Grapevine virus E (GVE, genus Vitivirus, family Betaflexiviridae) in vineyards in New York and California was conducted using macroarray hybridization or reverse-transcription polymerase chain reaction (RT-PCR) assays. In New York, GVE was detected in 10 of 46 vines of Vitis labrusca, one V. riparia, and one Vitis hybrid. All GVE-infected New York vines were coinfected with Grapevine leafroll-associated virus-3. In California, GVE was detected in 8 of 417 vines of V. vinifera. All GVE-infected California vines were also coinfected by one of the leafroll-associated viruses and other vitiviruses. In order to assess the genetic diversity among GVE isolates, a viral cDNA was amplified by RT-PCR, and a 675-nucleotide region that included the 3′ terminus of the coat protein gene, a short intergenic region, and the 5′ terminus of the putative nucleic acid binding protein gene was sequenced. All 20 GVE isolates sequenced in this study were very closely related, with >98% nucleotide identity to the SA94 isolate from South Africa. These findings confirm the presence of GVE in major grape-growing regions of the United States and indicate a very low level of genetic diversity.

Poxvirus infection in Hungarian great tits ( Parus major ): Case report

2008 ◽  
Vol 56 (4) ◽  
pp. 539-546 ◽  
Author(s):  
Elena Palade ◽  
Nóra Biró ◽  
Mihály Dobos-Kovács ◽  
Zoltán Demeter ◽  
Míra Mándoki ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Protein Gene ◽  
Core Protein ◽  
Parus Major ◽  
Great Tit ◽  
Nucleotide Sequence Analysis ◽  
Chain Reaction ◽  
Base Pairs ◽  
Polymerase Chain

From a total of 1819 great tits ( Parus major ) ringed in 2007 in Pilis Mountains, Hungary, 15 birds presented nodular proliferative lesions on different areas of the head and eyelids, suggesting a poxvirus infection. Three birds were submitted for analysis. The presence of avipoxvirus infection was confirmed by histopathology, electron microscopy (EM) and a polymerase chain reaction (PCR) based technique. Nucleotide sequence analysis of a 428 base pairs (bp) fragment of the viral 4b core protein gene revealed 100% identity between two of the Hungarian isolates (PM9 HUN, PM33 HUN) and two great tit poxvirus strains isolated in Norway in 1973 (GTV A256, GTV A311). The third Hungarian isolate (PM34 HUN) was more closely related to a different Norwegian isolate (GTVA310) than to the Hungarian isolates. The nucleotide sequence analysis of a shorter fragment of the viral 4b core protein (227 bp) gene revealed 100% identity between the Hungarian isolates, the same Norwegian isolates and a great tit poxvirus strain detected in Austria in 2007.

scholarly journals Polyvalent Degenerate Oligonucleotides Reverse Transcription-Polymerase Chain Reaction: A Polyvalent Detection and Characterization Tool for Trichoviruses, Capilloviruses, and Foveaviruses

2005 ◽  
Vol 95 (6) ◽  
pp. 617-625 ◽  
Author(s):  
Xavier Foissac ◽  
Laurence Svanella-Dumas ◽  
Pascal Gentit ◽  
Marie-Josée Dulucq ◽  
Armelle Marais ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Unknown Etiology ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Degenerate Primers ◽  
Sequence Comparisons ◽  
Pcr Products ◽  
Chlorotic Leaf ◽  
Polymerase Chain

A polyvalent nested reverse transcription-polymerase chain reaction (RT-PCR) test using degenerate primers containing inosine (polyvalent degenerate oligonucleotides [PDO]) was developed for filamentous fruit tree viruses belonging to the genera Trichovirus, Capillovirus, and Foveavirus. The 362-bp product was amplified from nucleic acid extracts obtained from Prunus and Malus leaf samples. All the viruses targeted were detected, demonstrating the polyvalence of the test. The variability of a collection of Apple chlorotic leaf spot virus isolates was analyzed using the sequence of the PDO RT-PCR amplified cDNAs. The technique was also used to screen stone fruit materials infected with known agents or with virus-like graft-transmissible diseases of unknown etiology. The results obtained further validated the broad specificity of the assay, with positive amplification obtained for uncharacterized or partially characterized viruses associated with cherry and peach disorders. Sequencing the amplified PCR products either directly or after cloning allowed the identification of variants of known agents and the tentative identification of two new agents, a Trichovirus and a Foveavirus. In addition, sequence comparisons demonstrated that the sequence of the targeted region is phylogenetically informative and of predictive taxonomic value.

scholarly journals Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant

2000 ◽  
Vol 124 (3) ◽  
pp. 481-487 ◽  
Author(s):  
P. J. MARKS ◽  
I. B. VIPOND ◽  
D. CARLISLE ◽  
D. DEAKIN ◽  
R. E. FEY ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Polymerase Chain Reaction ◽  
Inverse Relationship ◽  
Nucleotide Sequence Analysis ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Virus Particles ◽  
Stool Samples ◽  
Polymerase Chain

An outbreak of gastroenteritis followed a meal in a large hotel during which one of the diners vomited. The clinical features of the illness suggested Norwalk-like virus (NLV, small round structured virus) infection, and this was confirmed by electron microscopy and reverse transcriptase polymerase chain reaction (RT–PCR) of stool samples. Further characterization of the virus by nucleotide sequence analysis of the PCR amplicons revealed identical strains in all the affected individuals. The foods served at the meal could not be demonstrated to be the cause of the outbreak. Analysis of attack rates by dining table showed an inverse relationship with the distance from the person who vomited. No one eating in a separate restaurant reported illness. Transmission from person-to-person or direct contamination of food seems unlikely in this outbreak. However, the findings are consistent with airborne spread of NLV with infection by inhalation with subsequent ingestion of virus particles.

scholarly journals Investigating Infectious and Zoonotic diseases of Street Dogs in the Residential Area of Bangladesh Agricultural University

2015 ◽  
Vol 13 (1) ◽  
pp. 57-63
Author(s):  
TK Kasfi ◽  
SS Labony ◽  
MGA Chowdhury ◽  
UK Rima ◽  
MZ Hossain ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Matrix Protein ◽  
Protein Gene ◽  
Pcr Amplification ◽  
Zoonotic Diseases ◽  
Canine Distemper ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Matrix Protein Gene ◽  
Polymerase Chain

Dogs are common carnivor and scavenger at Bangladesh Agricultural University (BAU) campus. Dogs may carry many infectious and zoonotic diseases and could play a role in transmitting those diseases in other carnivors and human as well. At day time cats, dogs and golden jackals are found to eat in a same dustbin at BAU campus. So it is possible to share diseases of each other and there are possibilities of crossing species barrier. This study was, therefore, aimed at specific investigation of the occurrence of leishmaniasis, canine distemper (CD), infectious canine hepatitis (ICH) and avian influenza (AI) of dogs at BAU campus. This study was carried out during the period from January to June/ 2012. A total of 10 apparently healthy dogs were euthanatized and postmortem examination were carried out. Impression smears were prepared from spleen, liver, bone marrow and stained with Giemsa’s stain. Histopathological studies were conducted using routine procedures. For molecular characterization, polymerase chain reaction (PCR) and Reverse Transcriptase polymerase chain reaction (RT-PCR) were adopted for the detection of genomic fragment of visceral leishmaniasis (VL), canine distemper (CD), infectious canine hepatitis (ICH) and avian influenza (AI, viral matrix protein gene). Results of this study showed the presence of leishmania organism in the impression smears of eight dogs. Using PCR amplification technique, Leishmania donovani specific genomic DNA (145bp) was detected in two dogs. PCR amplification of genomic DNA of ICH (411bp) and RT-PCR amplification of genomic RNA of CD (287bp) and matrix protein gene of AI (245bp) were not detected in any of the dogs of BAU campus. Future work is needed to explore the presence of snad flies (vector of VL) in the investigating areas and establish role of street dogs of BAU campus towards disseminating leishmaniasis in other animals and human beings.DOI: http://dx.doi.org/10.3329/bjvm.v13i1.23720Bangl. J. Vet. Med. (2015). 13 (1): 57-63

scholarly journals Immunolocation of Aquaporin 8 in Human Cataractous Lenticular Epithelial Cells

2017 ◽  
Vol 2 (3) ◽  
pp. 1-5 ◽  
Author(s):  
Rijo Hayashi ◽  
Shimmin Hayashi ◽  
Kazunori Fukuda ◽  
Miki Sakai ◽  
Shigeki Machida
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Immunohistochemical Staining ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Anterior Capsule ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Anterior Capsulotomy ◽  
Density Results

Purpose/Aim: Aquaporin 8 (AQP8) is a diffusion facilitator of hydrogen peroxide (H2O2) through cell membranes. The purpose of this study was to confirm and localize AQP8 in human lenticular epithelial cells (LECs). Materials and Methods: Lenticular anterior capsule samples, including LECs, were collected during cataract surgery of cataract patients after informed consent. The localization of AQP8 was detected by immunohistochemical staining using an antibody to AQP8. Real-time polymerase chain reaction (RT-PCR) was also used to determine the AQP8 mRNA expression levels. The PCR products were analyzed by gel electrophoresis following analyses of band density. Results: Immunohistochemical staining showed AQP8 was distributed throughout the whole area of the anterior capsulotomy. AQP8 labeling was observed surrounding and within the cytoplasm of LECs. RT-PCR and gel electrophoresis also revealed the presence of AQP8 mRNA in the lenticular anterior capsule. The results of immunohistochemical staining were comparable to those of RT-PCR and gel electrophoresis. Conclusions: The results of this study indicate the distribution of AQP8 in human LECs. This is the first investigation confirming the presence of AQP8 in human LECs.

scholarly journals Nucleotide sequence of the coat protein gene of the Skierniewice isolate of plum pox virus (PPV).

1994 ◽  
Vol 41 (1) ◽  
pp. 87-95 ◽  
Author(s):  
K Wypijewski ◽  
T Malinowski ◽  
W Musiał ◽  
J Augustyniak
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Coat Protein ◽  
Amino Acid Sequence ◽  
Protein Gene ◽  
Plum Pox Virus ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Very High

The coat protein (CP) gene of the Skierniewice isolate of plum pox virus (PPV-S) has been amplified using the reverse transcription--polymerase chain reaction (RT-PCR), cloned and sequenced. The nucleotide sequence of the gene and the deduced amino-acid sequence of PPV-S CP were compared with those of other PPV strains. The nucleotide sequence showed very high homology to most of the published sequences. The motif: Asp-Ala-Gly (DAG), important for the aphid transmissibility, was present in the amino-acid sequence. Our isolate did not react in ELISA with monoclonal antibodies MAb06 supposed to be specific for PPV-D.

scholarly journals Keragaman sekuen DNA fragmen gen penyandi ACCase subunit BCCP dari tiga tipe kelapa sawit Variability of DNA sequence of gene fragment encoding BCCP subunit of ACCase from three types of oil palm

2016 ◽  
Vol 75 (1) ◽  
Author(s):  
Asmini BUDIANI ◽  
Djoko SANTOSO ◽  
A.R. PURBA PURBA
Keyword(s):  
Glycine Max ◽  
Oil Palm ◽  
Carrier Protein ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Pcr Products ◽  
Dna Fragment ◽  
Polymerase Chain

SummaryHeteromeric acetyl-CoA carboxylase (ht-ACCase) is one of key enzymes in palm oilbiosynthesis. Isolation and characterization ofthe gene is an important step in metabolicengineering to increase palm oil content andquality. The objective of this research was toisolate DNA fragment of gene encoding biotincarboxyl carrier protein (BCCP) subunit of ht-ACCase from three different oil palm types(Simalungun, Hibrida and Backcross) andinvestigate the variation of its DNA sequence.Total RNA was isolated from the mesocarp ofoil palm. DNA fragment encoding BCCP wasamplified by means of Reverse TranscriptasePolymerase Chain Reaction (RT-PCR) usingspecific primers with total RNA as a template.The products of RT-PCR were then purifiedfrom the gel, cloned and sequenced. The DNAsequences were analyzed for their homologiesto BCCP gene using BlastN and aligned todetect the sequence variability using ClustalWprogram from BioEdit. The results show thatone of the two RT-PCR products at about 300bp was highly homologous with the geneencoding BCCP from Glycine max, Brassicanapus and Arabidopsis thaliana. Nucleotidesequences of that BCCP fragments from thethree types of oil palm displayed some degreesof variability. Further investigation is neededto analyze the variability of the DNA sequencesof the full-length gene in relation with oilcontent or other characterRingkasanAsetil-CoA karboksilase heteromerik (ht-ACCase) merupakan salah satu enzim kuncidalam biosintesis minyak sawit. Isolasi dankarakterisasi gen tersebut merupakan langkahpenting dalam upaya rekayasa metabolismeuntuk peningkatan rendemen dan kualitasminyak sawit. Penelitian ini bertujuan untukmengisolasi fragmen DNA penyandi subunitbiotin carboxyl carrier protein (BCCP) dari ht-ACCase dari tiga tipe kelapa sawit yang ber-beda (Simalungun, Hibrida dan Backcross)dan mempelajari keragaman susunan nukleo-tidanya. RNA total diisolasi dari mesokarpbuah sawit. Fragmen gen penyandi BCCPdiamplifikasi dengan Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) meng-gunakan primer spesifik dan templat RNA total.Fragmen hasil RT-PCR dimurnikan dari gel,diklon kemudian disekuen. Sekuen DNA yangdiperoleh dianalisis homologinya dengan genBCCP menggunakan BlastN dan disejajarkanuntuk mengetahui keragamannya mengguna-kan program ClustalW dari BioEdit. Hasilnyamenunjukkan bahwa satu dari dua fragmenhasil RT-PCR yang berukuran sekitar 300 pbmemiliki homologi yang tinggi denganfragmen gen penyandi BCCP dari Glycine max,Brassica napus dan Arabidopsis thaliana.Urutan nukleotida fragmen BCCP dari ketigatipe kelapa sawit menunjukkan keragaman.Perlu analisis lebih lanjut mengenai keragamansekuen DNA dari gen lengkapnya dan dikajihubungannya dengan akumulasi minyak ataukarakter lain

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