Rapid identification of viruses causing sugarcane mosaic by direct sequencing of RT-PCR products from crude extracts: A method for large scale virus surveys

2009 ◽  
Vol 157 (2) ◽  
pp. 188-194 ◽  
Author(s):  
Maximiliano Gómez ◽  
Alejandro M. Rago ◽  
Germán Serino
2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Fahimeh Safarnezhad Tameshkel ◽  
Ali Salimi Jeda ◽  
Ahmad Tavakoli ◽  
Mohammad Hadi Karbalaie Niya ◽  
Morteza Izadi ◽  
...  

Background: Human rhinovirus (HRV) is still the most prevalent viral infection in humans and a significant cause of acute respiratory tract infections (ARTIs) in many communities, including military personnel undergoing basic training. Objectives: In this research, we assessed the molecular epidemiology, genotyping, and phylogenetic classification of HRVs in Iranian military trainees with respiratory infections (RI). Methods: For HRV identification and genotyping, respiratory specimens were obtained, and RT-PCR was conducted for genotyping and phylogenetic analysis of HRV utilizing primers for the 5-UTR region. Results: Among 400 Iranian military trainees (average age of 21 ± 4 years, the range of 18 - 57 years) with respiratory infections, HRV was detected in 29 patients (7%) using RT-PCR. The direct sequencing of PCR products from 10 specimens showed that the incidence of type A (n = 5, 50%) was higher than that of type B (n = 4, 40%) and type C (n = 1, 10%). There were no significant associations between HRV and respiratory and clinical symptoms, blood group, and indoor or outdoor conditions (P-value > 0.05). Conclusions: This research was the first to record HRV as a significant cause of respiratory problems among military trainees in Iran, with a frequency of 7%. The most prevalent genotype was HRV-A, which may be applicable in epidemiological and clinical studies, as well as vaccination plans.


1996 ◽  
Vol 270 (3) ◽  
pp. C885-C891 ◽  
Author(s):  
R. J. Hughes ◽  
K. L. Anderson ◽  
D. Kiel ◽  
P. A. Insel

Beta-adrenergic receptor kinase is a member of the G protein-linked receptor kinase (GRK1) family that elicits receptor desensitization. We have cloned GRK2 from S49 mouse lymphoma cells. The nucleotide sequences of rat GRK2 and GRK3 were aligned and conserved primers chosen for use in reverse transcription-polymerase chain reaction (RT-PCR) of S49 mRNA. Direct sequencing of the PCR fragment provided a rapid means to identify the expression of the GRK2 but not the GRK3 transcript in these cells. Unique expression of GRK2 in S49 cells was confirmed by Western blotting. Three additional pairs of primers were chosen from the rat GRK2 sequence to amplify overlapping regions that together encompassed the entire coding sequence. After attempts to ligate the four fragments of S49 cell GRK2 cDNA by using PCR proved unsuccessful, the intact cDNA was assembled by digesting the PCR products in the region of the overlaps and ligating them in a single step into pBlue-script SK(+).


Plant Disease ◽  
2015 ◽  
Vol 99 (2) ◽  
pp. 293-293 ◽  
Author(s):  
M. Beuve ◽  
T. Candresse ◽  
M. Tannières ◽  
O. Lemaire

Grapevine Pinot gris virus (GPGV), belonging to the genus Trichovirus of the family Betaflexiviridae, was first identified by siRNA sequencing in northern Italy in 2012, in the grapevine varieties Pinot gris, Traminer, and Pinot Noir, which exhibited mottling and leaf deformation (1), and in asymptomatic vines, with a lower frequency. Since 2012, this virus has also been reported in South Korea, Slovenia, Greece (3), Czech Republic (2), Slovakia (2), and southern Italy (4). In 2014, GPGV was identified by Illumina sequencing of total RNAs extracted from leaves of the Merlot variety (Vitis vinifera) grafted onto Gravesac rootstock originated from a vineyard in the Bordeaux region of France. This Merlot plant exhibited fanleaf-like degeneration symptoms associated with Tomato black ring virus (TBRV) infection. Cuttings were collected in 2010 and maintained thereafter in a greenhouse. The full-length genome was assembled either de novo or by mapping of the Illumina reads on a reference GPGV genome (GenBank FR877530) using the CLC Genomics workbench software (CLC Bio, Qiagen, USA). The French GPGV isolate “Mer” (7,223 nucleotides, GenBank KM491305) is closely related to other European GPGV sequences; it exhibits 95.4% nucleotide identity with the reference Italian isolate (NC_015782) and 98 to 98.3% identity with Slovak isolates (KF134123 to KF134125). The higher divergence between French and Italian GPGV isolates was mainly due to differences in the 5′ extremity of the genome, as already shown with the Slovak GPGV isolates. RNA extracted from phloem scrapings of 19 cv. Merlot vines from the same plot collected in 2014 were analyzed by RT-PCR using the specific primer pair Pg-Mer-F1 (5′-GGAGTTGCCTTCGTTTACGA-3′) and Pg-Mer-R1 (5′-GTACTTGATTCGCCTC GCTCA-3′), designed on the basis of alignments of all available GPGV sequences from GenBank. The resulting amplicon of 770 bp corresponded to a fragment of the putative movement protein (MP) gene. Seven (35%) of the tested plants gave a strong positive amplification. Three RT-PCR products were directly sequenced and showed 99.3 to 99.5% identity within the MP gene of the GPGV-Mer isolate. Given the mixed viral infection status of the vines found infected by GPGV, it was not possible to associate a specific symptomatology with the presence of GPGV. Furthermore, similar RT-PCR tests were also performed on RNA extracts prepared from two plants of cv. Carignan that originated from a French grapevine collection, exhibiting fanleaf-like symptoms without any nepovirus detection. These samples similarly gave a strong positive amplification. The sequences obtained from the two Carignan vines showed 98.4 and 97.8% identity with the GPGV-Mer isolate. To our knowledge, this is the first report of GPGV in France. GPGV has been discovered in white and red berry cultivars, suggesting that its prevalence could be important in European vineyards (2). Further large-scale studies will be essential to determine the world prevalence of GPGV and to evaluate its potential effects on yield and on wine quality, as well as to shed light on GPGV epidemiology. Of particular concern is whether, like the other grapevine-infecting Trichovirus, Grapevine berry inner necrosis virus (GPGV) can be transmitted by the eryophid mite Colomerus vitis. References: (1) A. Giampetruzzi et al. Virus Res. 163: 262, 2012. (2) M. Glasa et al. Arch. Virol. 159: 2103, 2014. (3) G. P. Martelli, J. Plant Pathol. 96: S105, 2014. (4) M. Morelli et al. J. Plant Pathol. 96:431, 2014.


Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1310
Author(s):  
Ivan Toplak ◽  
Laura Šimenc ◽  
Metka Pislak Ocepek ◽  
Danilo Bevk

In recent years, there has been growing evidence that certain types of honeybee viruses could be transmitted between different pollinators. Within a voluntary monitoring programme, 180 honeybee samples (Apis mellifera carnica) were collected from affected apiaries between 2007 and 2018. Also from August 2017 to August 2018, a total 148 samples of healthy bumblebees (Bombus lapidarius, B. pascuorum, B. terrestris, B. lucorum, B. hortorum, B. sylvarum, B. humilis) were collected at four different locations in Slovenia, and all samples were tested by using RT-PCR methods for six honeybee viruses. Direct sequencing of a total 158 positive samples (acute bee paralysis virus (ABPV n = 33), black queen cell virus (BQCV n = 75), sacbrood bee virus (SBV n = 25) and Lake Sinai virus (LSV n = 25)) was performed from obtained RT-PCR products. The genetic comparison of identified positive samples of bumblebees and detected honeybee field strains of ABPV, BQCV, SBV, and LSV demonstrated 98.74% to 100% nucleotide identity between both species. This study not only provides evidence that honeybees and bumblebees are infected with genetically identical or closely related viral strains of four endemically present honeybee viruses but also detected a high diversity of circulating strains in bumblebees, similar as was observed among honeybees. Important new genetic data for endemic strains circulating in honeybees and bumblebees in Slovenia are presented.


Plant Disease ◽  
2011 ◽  
Vol 95 (10) ◽  
pp. 1321-1321 ◽  
Author(s):  
K. Hamed ◽  
W. Menzel ◽  
G. Dafalla ◽  
A. M. A. Gadelseed ◽  
S. Winter

In summer 2009, a survey for virus diseases in cucurbits was conducted in open fields and plastichouses in Khartoum State, the most important growing area for cucurbits in Sudan. Chlorosis and yellowing symptoms on middle and lower leaves were observed on many muskmelon (Cucumis melo L.) plants grown in open fields in the Assilat agricultural scheme and on approximately 80% of the cucumber (Cucumis sativus L.) plants grown in plastichouses in Khartoum North. Large populations of whiteflies (Bemisia tabaci L.) were present in both locations. Leaf symptoms that were observed were similar to those caused by Cucurbit chlorotic yellows virus (CCYV), a recently described new Crinivirus species infecting cucurbits in Japan (4), indicating presence of this virus previously only reported from Japan, Taiwan (2), and China (1). Samples from seven symptomatic muskmelon leaves were collected from individual plants grown in different open fields in Assilat and from a symptomatic cucumber plant grown in a plastichouse. Total RNA was extracted from these samples with the RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany) to amplify putative CCYV sequences with primers (Crini-s2 5′-CATTCCTACCTGTTTAGCCA and Crini-as2 5′-TGCACTTATAATCTGCTGGTAC) designed from CCYV sequences available at GenBank. A 353-bp DNA fragment of the HSP70 gene was amplified by reverse transcription (RT)-PCR for all samples. Further analysis by direct sequencing of two PCR products showed 99 to 100% nt sequence identity to Asian CCYV isolates. Amplification of the coat protein sequence with the primer pair (CCYV-CPs 5′-ATGGAGAAGACTGACAATAAACAA and CCYV-CPas 5′-TTTACTACAACCTCCCGGTG) followed by cloning and sequencing yielded a 760-bp fragment having 99% nucleotide sequence identity to all Asian isolates. For confirmation, dsRNA preparations of symptomatic muskmelon tissue (collected in June 2010) were made, showing dsRNA patterns typical for criniviruses after separation on agarose gels. This dsRNA was used as template for random RT-PCR followed by sequencing of the cloned PCR products (3). Comparison with sequences available at GenBank revealed that cDNA sequences from dsRNA also were 99 to 100% identical to the CCYV genome sequence (AB523788.1). Whitefly transmission of the virus was confirmed by giving a population of B. tabaci an acquisition access period of 24 h and a further 24 h on muskmelon and cucumber seedlings. Symptoms were observed after 5 to 7 days, and the presence of CCYV was confirmed by RT-PCR. In conclusion, symptoms, RT-PCR, and dsRNA sequencing results confirm the presence and establishment of CCYV in cucurbit crops in Sudan. It is remarkable that the sequences obtained from the Sudanese samples show only negligible sequence differences from Asian isolates. Because of the large whitefly vector populations, the spread of CCYV to neighboring countries in Africa and potentially southern Europe, or wherever cucurbits are grown, can be expected. To our knowledge, this is the first report of CCYV in Sudan and outside Eastern Asia. The sequences obtained in this study have been submitted to GenBank under Accession Nos. JF807053 to JF807055. References: (1) Q. S. Gu et al. Plant Dis. 95:73, 2011. (2) L. H. Huang et al. Plant Dis. 94:1168, 2010. (3) W. Menzel et al. Arch. Virol. 154:1343, 2009. (4) M. Okuda et al. Phytopathology 100:560, 2010.


2020 ◽  
Vol 2020 ◽  
pp. 1-5
Author(s):  
Catia Mio ◽  
Adriana Cifù ◽  
Stefania Marzinotto ◽  
Natascha Bergamin ◽  
Chiara Caldana ◽  
...  

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has rapidly spread worldwide from the beginning of 2020. The presence of viral RNA in samples by nucleic acid (NA) molecular analysis is the only method available to diagnose COVID-19 disease and to assess patients’ viral load. Since the demand for laboratory reagents has increased, there has been a worldwide shortage of RNA extraction kits. We, therefore, developed a fast and cost-effective viral genome isolation method that, combined with quantitative RT-PCR assay, detects SARS-CoV-2 RNA in patient samples. The method relies on the addition of Proteinase K followed by a controlled heat-shock incubation and, then, E gene evaluation by RT-qPCR. It was validated for sensitivity, specificity, linearity, reproducibility, and precision. It detects as low as 10 viral copies/sample, is rapid, and has been characterized in 60 COVID-19-infected patients. Compared to automated extraction methods, our pretreatment guarantees the same positivity rate with the advantage of shortening the time of the analysis and reducing its cost. This is a rapid workflow meant to aid the healthcare system in the rapid identification of infected patients, such as during a pathogen-related outbreak. For its intrinsic characteristics, this workflow is suitable for large-scale screenings.


2014 ◽  
Vol 15 (3) ◽  
pp. 122-123 ◽  
Author(s):  
Jang-Kyun Seo ◽  
Ja-Yeon Park ◽  
Hae-Ryun Kwak ◽  
Mi-Kyeong Kim ◽  
Moon Nam ◽  
...  

To determine the current incidence of Soybean yellow mottle mosaic virus (SYMMV), Soybean yellow common mosaic virus (SYCMV), and Soybean mosaic virus (SMV) in Korea, a nationwide survey was performed in 2013. A total of 417 soybean samples with typical virus symptoms were collected from 9 large-scale soybean-cultivated areas. To identify viruses from the collected samples, RT-PCR was performed using specific primers for SMV, SYMMV, or SYCMV. To confirm the RT-PCR results, PCR products were purified and sequenced. 22 July 2014. 18 August 2014.


2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e20017-e20017 ◽  
Author(s):  
Y. Zhu ◽  
L. Si ◽  
Y. Kong ◽  
Z. Chi ◽  
X. Yuan ◽  
...  

e20017 Background: To investigate the possible relationship between sunitinib response and KIT mutation as well as KIT expression. Methods: We find that a 75-year male patient with KIT-mutated metastatic mucosal (primary: nasal, metastasis: left adrenal gland, lungs and right eye) melanoma has a very good response to sunitinib 37.5mg/d consecutively. We apply IHC sp assay to detect S100, HMB-45, CD117, VEGFR, PDGFR and EGFR in the melanoma sample of this patient. DNA is also extracted from paraffin-embedded melanoma sample of this patient and PCR is carried out to amplify several exons of C-KIT (exon11,13,17,18),B-RAF(exon11, 15) and N- RAS(exon1, 2). PCR products are sent to be directly sequenced. Clinical response, PFS and OS of the patient are evaluated. Results: Strong expression of S100, HMB45, CD117, VEFGR and PDGFR is detected by IHC in the tissue sample of the patient. Furthermore, direct sequencing after PCR reveals that V559A substitution is detected in exon 11 of KIT, with mutation of T into C at 1776, leading to substitution of Valine by Alanine at 559. No mutation was detected in exon 9, 13, 17, 18 of KIT, B-RAF15, 11 or N-RAS1, 2. The patient received sunitinib 37.5mg/d from July 2008 consecutively and evaluated PR (Recist: tumors shrink 70%) lasting for 5 months with Grade II myelosuppression and stomatitis. Now the patient is still alive and received sunitinib. Conclusions: Our case report suggests that response of melanoma patients to sunitinib might be correlated to KIT mutation and expression, and large-scale clinical trial in patients with KIT mutation and over-expression might be useful to shed light on treatment of melanoma patients with individual targeted therapy. No significant financial relationships to disclose.


2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).


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