UTR Sequencing Research Articles

Genotypic analysis of a localised hotspot of Pestivirus A (BVDV-1) infections in Northern Ireland.

Bovine viral diarrhoea (BVD) is caused by Pestivirus A and Pestivirus B. Northern Ireland (NI) embarkedon a compulsory BVD eradication scheme in 2016, which continues to this day, so an understanding of the composition of the pestivirus genotypes in the cattle population of NI is required. This molecular epidemiology study employed 5' untranslated region (5'UTR) genetic sequencing to examine the pestivirus genotypes circulating in samples taken from a hotspot of BVD outbreaks in the Enniskillen area in 2019. Bovine viral diarrhoea virus (BVDV)-1e (Pestivirus A) was detected for the first time in Northern Ireland, and at a highfrequency, in an infection hotspot in Enniskillen in 2019. There was no evidence of infection with BVDV-2 (Pestivirus B), Border disease virus (pestivirus D) or HoBi-like virus/BVDV-3 (pestivirus H). Only 5'UTR sequencing was used, so supplementary sequencing, along with phylogenetic trees that include all BVDV-1 genotype reference strains, would improve accuracy. Examination of farm locations and animal movement/trade is also required. Genotype BVDV-1e was found for the first time in Northern Ireland, indicating an increase in the genetic diversity of BVDV-1, which could have implications for vaccine design and highlights the need for continued pestivirus genotypic surveillance.

T helper cells exhibit a dynamic and reversible 3'-UTR landscape.

3' untranslated regions (3' UTRs) are critical elements of messenger RNAs, as they contain binding sites for RNA-binding proteins (RBPs) and microRNAs that affect various aspects of the RNA life cycle including transcript stability and cellular localization. In response to T cell receptor activation, T cells undergo massive expansion during the effector phase of the immune response and dynamically modify their 3' UTRs. Whether this serves to directly regulate the abundance of specific mRNAs or is a secondary effect of proliferation remains unclear. To study 3'-UTR dynamics in T helper cells, we investigated division-dependent alternative polyadenylation (APA). In addition, we generated 3' end UTR sequencing data from naive, activated, memory, and regulatory CD4+ T cells. 3'-UTR length changes were estimated using a nonnegative matrix factorization approach and were compared with those inferred from long-read PacBio sequencing. We found that APA events were transient and reverted after effector phase expansion. Using an orthogonal bulk RNA-seq data set, we did not find evidence of APA association with differential gene expression or transcript usage, indicating that APA has only a marginal effect on transcript abundance. 3'-UTR sequence analysis revealed conserved binding sites for T cell-relevant microRNAs and RBPs in the alternative 3' UTRs. These results indicate that poly(A) site usage could play an important role in the control of cell fate decisions and homeostasis.

Diversity of Human Enterovirus Co-Circulations in Five Kindergartens in Bangkok between July 2019 and January 2020.

Human enterovirus causes various clinical manifestations in the form of rashes, febrile illness, flu-like illness, uveitis, hand-foot-mouth disease (HFMD), herpangina, meningitis, and encephalitis. Enterovirus A71 and coxsackievirus are significant causes of epidemic HFMD worldwide, especially in children aged from birth to five years old. The enterovirus genotype variants causing HFMD epidemics have been reported increasingly worldwide in the last decade. We aim to use simple and robust molecular tools to investigate human enteroviruses circulating among kindergarten students at genotype and subgenotype levels. With the partial 5'-UTR sequencing analysis as a low-resolution preliminary grouping tool, ten enterovirus A71 (EV-A71) and coxsackievirus clusters were identified among 18 symptomatic cases and 14 asymptomatic cases in five kindergartens in Bangkok, Thailand, between July 2019 and January 2020. Two occurrences of a single clone causing an infection cluster were identified (EV-A71 C1-like subgenotype and coxsackievirus A6). Random amplification-based sequencing using MinION (Oxford Nanopore Technology) helped identify viral transmission between two closely related clones. Diverse genotypes co-circulating among children in kindergartens are reservoirs for new genotype variants emerging, which might be more virulent or better at immune escape. Surveillance of highly contagious enterovirus in communities is essential for disease notifications and controls.

ADAR1p110 promotes Enterovirus D68 replication through its deaminase domain and inhibition of PKR pathway

BackgroundSevere respiratory and neurological diseases caused by human enterovirus D68 (EV-D68) pose a serious threat to public health, and there are currently no effective drugs and vaccines. Adenosine deaminase acting on RNA1 (ADAR1) has diverse biological functions in various viral infections, but its role in EV-D68 infections remains undetermined.MethodsRhabdomyosarcoma (RD) and human embryonic kidney 293 T (293 T) cells, and HeLa cells were used to evaluate the expression level of ADAR1 upon EV-D68 (Fermon strain) and human parainfluenza virus type 3 (HPIV3; NIH47885) infection, respectively. Knockdown through silencing RNA (siRNA) and overexpression of either ADAR1p110 or ADAR1p150 in cells were used to determine the function of the two proteins after viral infection. ADAR1p110 double-stranded RNA binding domains (dsRBDs) deletion mutation was generated using a seamless clone kit. The expression of ADAR1, EV-D68 VP1, and HPIV3 hemagglutinin–neuraminidase (HN) proteins was identified using western blotting. The median tissue culture infectious dose (TCID50) was applied to detect viral titers. The transcription level of EV-D68 mRNA was analyzed using reverse transcription-quantitative PCR (RT-qPCR) and the viral 5′-untranslated region (5′-UTR)-mediated translation was analyzed using a dual luciferase reporter system.ConclusionWe found that the transcription and expression of ADAR1 was inhibited upon EV-D68 infection. RNA interference of endogenous ADAR1 decreased VP1 protein expression and viral titers, while overexpression of ADAR1p110, but not ADAR1p150, facilitated viral replication. Immunofluorescence assays showed that ADAR1p110 migrated from the nucleus to the cytoplasm after EV-D68 infection. Further, ADAR1p110 lost its pro-viral ability after mutations of the active sites in the deaminase domain, and 5′-UTR sequencing of the viral genome revealed that ADAR1p110 likely plays a role in EV-D68 RNA editing. In addition, after ADAR1 knockdown, the levels of both phosphorylated double-stranded RNA dependent protein kinase (p-PKR) and phosphorylated eukaryotic initiation factor 2α (p-eIF2α) increased. Attenuated translation activity of the viral genome 5′-UTR was also observed in the dual-luciferase reporter assay. Lastly, the deletion of ADAR1p110 dsRBDs increased the level of p-PKR, which correlated with a decreased VP1 expression, indicating that the promotion of EV-D68 replication by ADAR1p110 is also related to the inhibition of PKR activation by its dsRBDs. Our study illustrates that ADAR1p110 is a novel pro-viral factor of EV-D68 replication and provides a theoretical basis for EV-D68 antiviral research.

Comparison of Sanger sequencing for hepatitis C virus genotyping with a commercial line probe assay in a tertiary hospital

BackgroundThe technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5’UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results.Methods100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5’UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank.ResultsAll the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5’UTR and Core. Twenty-three samples were sequenced for two regions: 5′ UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5’UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%.ConclusionsIn this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.

Hepatitis C virus transmission in a Dutch haemodialysis unit: detailed outbreak investigation using NS5A gene sequencing

Haemodialysis is a risk factor for hepatitis C virus (HCV) transmission. Two patients receiving haemodialysis in a Dutch dialysis unit in The Hague were found to seroconvertto HCV in December 2016 after the yearly routine control for blood-borne viruses. Following the presumed time of infection, three chronically infected HCV patients were identified as possible index cases. To confirm inter-patient transmission and to identify the source. Molecular investigation and review of medical records were performed. Both of the incident cases and one of the three possible index cases were demonstrated to be infected with HCV genotype 2b based on 5'UTR sequencing. Epidemiological relatedness between these viruses was further investigated by sequencing of the NS5A region. Phylogenetic analysis clearly identified the incident cases and the index case to represent a cluster distinct from unrelated controls with HCV genotype 2b. Detailed review of the medical records identified two possible incidents that might have resulted in the HCV transmission cases: contamination of the venous pressure-sensing port due to high venous pressures or incomplete compliance with infection control precautions of the unit staff during handling of two incidents, that occurred at the same time in a single haemodialysis session with the index patient as well as both incident cases present. This study demonstrates that detailed incident recording in combination with state-of-the-art molecular investigations such as sequencing of the NS5A region resulted in unravelling a set of two HCV transmissions that occurred at a haemodialysis unit.

Molecular detection and characterization of transient bovine viral diarrhea virus (BVDV) infections in cattle commingled with ten BVDV persistently infected cattle.

Fifty-three cattle of unknown serologic status that were not persistently infected (PI) with bovine viral diarrhea virus (BVDV) were commingled with 10 cattle that were PI with different strains of BVDV, and were monitored for an extended commingle period using a reverse-transcription real-time PCR (RT-rtPCR) BVDV assay on various sample types. Transient infections with BVDV were also assessed by virus isolation, virus neutralization (VN) assays, and direct buffy coat 5'-UTR sequencing. Infections were demonstrated in all cattle by RT-rtPCR; however, the detection rate was dependent on the type of sample. Buffy coat samples demonstrated a significantly greater number of positive results ( p ≤ 0.05) than either serum or nasal swab samples. Presence of elevated BVDV VN titers at the onset inversely correlated with the number of test days positive that an individual would be identified by RT-rtPCR from buffy coat samples, and directly correlated with the average Ct values accumulated over all RT-rtPCR test days from buffy coat samples. Both single and mixed genotype/subgenotype/strain infections were detected in individual cattle by direct sample 5'-UTR sequencing. A BVDV-2a strain from a PI animal was found to be the predominant strain infecting 64% of all non-PI cattle; BVDV-1b strains originating from 3 PI cattle were never detected in non-PI cattle. Although direct sample 5'-UTR sequencing was capable of demonstrating mixed BVDV infections, identifying all strains suspected was not always efficient or possible.

FRI-178 - Evaluation of a sequence-based method for hepatitis C genotyping (Deepchek® SingleRound PCR and NS5B/5’UTR sequencing) and comparison with VERSANT HCV Genotype 2.0 assay (InnoLiPA)

FRI-178 - Evaluation of a sequence-based method for hepatitis C genotyping (Deepchek® SingleRound PCR and NS5B/5’UTR sequencing) and comparison with VERSANT HCV Genotype 2.0 assay (InnoLiPA)

Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5' Untranslated Region (5'UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping.

Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100%. The highest prevalent genotype was 1b (50%) followed by genotypes 1a (16%), 4a (12%), 2a (10%), 2c (8%), and 3a (4%). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations.

CisSERS: Customizable In Silico Sequence Evaluation for Restriction Sites.

High-throughput sequencing continues to produce an immense volume of information that is processed and assembled into mature sequence data. Data analysis tools are urgently needed that leverage the embedded DNA sequence polymorphisms and consequent changes to restriction sites or sequence motifs in a high-throughput manner to enable biological experimentation. CisSERS was developed as a standalone open source tool to analyze sequence datasets and provide biologists with individual or comparative genome organization information in terms of presence and frequency of patterns or motifs such as restriction enzymes. Predicted agarose gel visualization of the custom analyses results was also integrated to enhance the usefulness of the software. CisSERS offers several novel functionalities, such as handling of large and multiple datasets in parallel, multiple restriction enzyme site detection and custom motif detection features, which are seamlessly integrated with real time agarose gel visualization. Using a simple fasta-formatted file as input, CisSERS utilizes the REBASE enzyme database. Results from CisSERS enable the user to make decisions for designing genotyping by sequencing experiments, reduced representation sequencing, 3’UTR sequencing, and cleaved amplified polymorphic sequence (CAPS) molecular markers for large sample sets. CisSERS is a java based graphical user interface built around a perl backbone. Several of the applications of CisSERS including CAPS molecular marker development were successfully validated using wet-lab experimentation. Here, we present the tool CisSERS and results from in-silico and corresponding wet-lab analyses demonstrating that CisSERS is a technology platform solution that facilitates efficient data utilization in genomics and genetics studies.

Global 3' UTR Sequencing in Multiple Myeloma (MM)

The 3' untranslated region (3'UTR) of mRNA contains elements that play regulatory roles in polyadenylation, localization, translation efficiency, and mRNA stability. Although relative contributions of different regulatory mechanisms remain poorly understood, several recent studies showed that alterations in 3'UTRs may affect protein localization as well as their interactions with other proteins.Here, we obtained global measurements of 3'UTR sequencing and RNA-seq in MM and normal plasma cells to study the effects of 3'UTR alterations in MM. The transcriptomes of 15 MM samples and two samples from normal plasma cells were assessed by RNA-seq and alternative 3'UTR isoforms were measured using 3'-seq, an established quantitative 3' end sequencing method.We first studied gene expression results from two different platforms to estimate the overlap of mRNA abundance. For each individual sample there was a high level of correlation between 3'-seq and RNA-seq in terms of identifying genes that were up or down regulated. We identified a common set of 195 genes that were significantly different between normal plasma cells and the 15 MM samples as measured with two independent platforms and this list was enriched in genes regulated by NF-kB in response to TNF and genes up-regulated by KRAS activation pathways.In addition to finding differences in mRNA abundance levels, with 3'-seq, we found 187 genes that had shorter 3'UTRs and 170 genes that had longer 3'UTRs in MM compared to normal plasma cells. Genes that had shorter 3'UTR sequences were enriched for those localized to nucleus and endoplasmic reticulum. They were also enriched in RNA metabolic processes, RNA splicing as well as DNA repair. Genes that had longer 3'UTRs were enriched for genes involved in signal transduction affecting a change in the level or activity of a second messenger or other downstream targets, and ultimately affecting a change in cellular functions. We observed enrichment for G2/M checkpoint, as in progression through the cell division cycle and activation of mTORC1 complex and MYC targets for both shorter and longer 3'UTR genes, which suggests we should investigate these genes deeply to understand their individual regulatory roles in MM development. We found five oncogenes that had shorter 3'UTRs, whereas genes with longer 3'UTRs contained two oncogenes and two tumor suppressor genes.Interestingly amongst 15 patients, five patients had UTR pattern similar to normal plasma cells, 6 had higher frequency of longer UTRs and 4 patients had relatively shorter UTRs compared to normal plasma cells. This pattern did not correlate with common cytogenetic abnormalities. Further investigation is now required to understand the functional consequences of UTR changes and to correlate clinical and prognostic impact of such UTR differences. In conclusion, we report that significant 3'UTR alterations are observed in MM with impact on MM cell pathobiology and suggesting need for further investigation to understand their precise role in regulation and translation of proteins in MM. DisclosuresMunshi:millenium: Membership on an entity's Board of Directors or advisory committees; onyx: Membership on an entity's Board of Directors or advisory committees; novartis: Membership on an entity's Board of Directors or advisory committees; celgene: Membership on an entity's Board of Directors or advisory committees.

Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China.

To determine the nationwide status of persistent BVDV infection in different bovine species in China and compare different test methods, a total of 1379 serum samples from clinical healthy dairy cattle, beef cattle, yaks (Bos grunniens), and water buffalo (Bubalus bubalis) were collected in eight provinces of China from 2010 to 2013. The samples were analyzed using commercial antibody (Ab) and antigen (Ag) detection kits, and RT-PCR based on the 5’-UTR and Npro gene sequencing. Results showed that the overall positive rates for BVDV Ab, Ag and RT-PCR detection were 58.09% (801/1379), 1.39% (14/1010), and 22.64% (146/645), respectively, while the individual positive rates varied among regions, species, and farms. The average Ab-positive rates for dairy cattle, beef cattle, yaks, and water buffalo were 89.49% (298/333), 63.27% (248/392), 45.38% (236/520), and 14.18% (19/134), respectively, while the Ag-positive rates were 0.00% (0/116), 0.77% (3/392), 0.82% (3/368), and 5.97% (8/134), respectively, and the nucleic acid-positive rates detected by RT-PCR were 32.06% (42/131), 13.00% (26/200), 28.89% (52/180), and 19.40% (26/134), respectively. In addition, the RT-PCR products were sequenced and 124 5’-UTR sequences were obtained. Phylogenetic analysis of the 5’-UTR sequences indicated that all of the 124 BVDV-positive samples were BVDV-1 and subtyped into either BVDV-1b (33.06%), BVDV-1m (49.19%), or a new cluster, designated as BVDV-1u (17.74%). Phylogenetic analysis based on Npro sequences confirmed this novel subtype. In conclusion, this study revealed the prevalence of BVDV-1 in bovine species in China and the dominant subtypes. The high proportion of bovines with detectable viral nucleic acids in the sera, even in the presence of high Ab levels, revealed a serious threat to bovine health.

Evaluation of the Abbott RealTime HCV genotype II RUO (GT II) assay with reference to 5′UTR, core and NS5B sequencing

BackgroundHCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. ObjectiveTo evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5′UTR, core and NS5B. Study designThe Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5′UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. ResultsAmong routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5′UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5′UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. ConclusionsThe Abbott assay is an automated HCV genotyping method with improved accuracy over 5′UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method.

Naturally occurring hepatitis C virus (HCV) NS3/4A protease inhibitor resistance-related mutations in HCV genotype 1-infected subjects in Italy

To assess the prevalence of hepatitis C virus (HCV) NS3/4A protease inhibitor (PI) resistance mutations in HCV genotype 1-infected PI-naive individuals in Italy. One hundred and twelve patients infected with HCV genotype 1a or 1b (based on Versant HCV Genotype 2.0 or 5'UTR/core sequencing) and never treated with any HCV PI were evaluated. The whole NS3 region was analysed by population sequencing and mutations related to resistance to linear and macrocyclic PIs were recorded. Forty-six HCV-monoinfected and 66 HCV/HIV-coinfected subjects were studied. Complete NS3 sequence information was obtained for 109 (97.3%) samples: 67 subtype 1a and 42 subtype 1b. Subtype assignment by NS3 sequencing was concordant in 100.0% and 83.9% of cases with the original 5'UTR sequencing and Versant result, respectively. At least one mutation related to PI resistance was detected in 21 (19.3%) isolates. However, 11 of these had only Q80K, expected to confer resistance to one investigational macrocyclic compound, and were detected only in subtype 1a. Boceprevir and telaprevir resistance-related mutations were detected in 10 (9.2%) isolates and included V36L, T54S and V55A. Only one isolate harboured two mutations (V36L and T54S). There was no association between HCV PI resistance and HIV coinfection or exposure to HIV PIs. A minority of untreated HCV genotype 1 patients in Italy harbour a virus population carrying HCV PI resistance-related mutations. The clinical implications of this finding warrant further analysis.

Changing HCV genotypes distribution in Poland—Relation to source and time of infection

Background Understanding the distribution of HCV genotypes has implications for prognosis and therapy of hepatitis C. Objectives To describe the distribution of HCV genotypes in Poland in relation to route of transmission and year of infection. Study design Patients with chronic liver disease were evaluated at the Department of Infectious Diseases, Bialystok (Poland). HCV genotype was determined by means of 5′UTR sequencing and comparison with known sequences of particular genotypes. Results The genotypes mostly frequently detected were genotype 1 (57.5%); genotype 3 (31.3%); and genotype 4 (8.4%). Genotype 1 constituted the majority of HCV infections caused by blood transfusion (68.8%) and only 34.8% of HCV infections in the intravenous drug use (IVDU) group ( p < 0.05). In contrast genotype 3 constituted the majority of HCV infections in the IVDU group (56.5%). We observed a significant increase in the proportion of genotype 3 infections detected after 2000—from 19.1% to 38.9%. Conclusions The relative proportion of genotype 1b in Poland has decreased and that of genotype 3a has increased, especially among IVDU.

High prevalence of genotype 4 among hepatitis C virus‐infected intravenous drug users in North‐Eastern Poland

The aim of this study was to describe the distribution of hepatitis C genotypes among intravenous drug users in north-eastern Poland. The study group included intravenous drug users recruited at a drug treatment center and a clinic for HIV-infected patients. HCV infection was confirmed by qualitative nested RT-PCR to test for the presence of HCV RNA. Genotypes were determined by 5'UTR sequencing and comparing the results with known genotype sequences. Among 111 HCV-infected and HCV-RNA-positive intravenous drug users, the most prevalent genotypes were 1 (38.7%), 3 (37.8%), and 4 (23.4%). Most infections with genotype 4 (88.5%) were found among HCV-HIV-coinfected drug users. The study demonstrated a high prevalence of genotype 4 (23.4%) among HCV-infected Polish drug users.

Ruolo del sequenziamento diretto nella tipizzazione di HCV-RNA a fini clinico-epidemiologici

Background/Aims: Genotyping and subtyping of hepatitis C virus (HCV) is epidemiologically and clinically relevant to prognosis and therapeutical management of HCV infection.Aim was to study the feasibility of an “in house” direct sequencing method for the genotyping and subtyping of HCV strains compared with a commercially available genotyping assay (Inno-LiPA,VERSANT® HCV Genotype 2.0 Assay, Bayer). Methods: 74 clinical plasma samples cross-representing HCV genotypes 1 to 5 typed with the line probe assay Inno-LiPA (LiPA) were subjected to a laboratory-developed 5’UTR direct sequencing protocol (5’UTR Seq).A NS5B direct sequencing protocol (NS5B Seq) was apply to 23/74 samples (17 negative by the 5’UTR).Two libraries of 5’UTR and NS5B HCV prototypes were constructed; BioEdit 7.0.0 and ClustalW were used for alignments and phylogenetic analysis. Results: 5’UTR Seq typed 47/74 LiPA positive samples (64%). Concordance between the two assays was 91% (43/47) and 72% (31/43) for HCV genotyping and subtyping, respectively. LiPA and 5’UTR Seq discordant samples were typed by NS5B Seq. NS5B Seq typed 13/17 samples negative with 5’UTR Seq.All genotypes and subtypes detected with NS5B Seq were concordant with LiPA. 5’UTR and NS5B direct sequencing pointed out a wider HCV subtype distribution than LiPA for genotype 1, 2 and 4. By the combination of 5’UTR and NS5B direct sequencing, 81% (60/74) of LiPA positive samples were typed. Conclusion: HCV typing and subtyping with the line probe assay Inno-LiPA is highly recommended for clinical purposes.A more detailed HCV typing for epidemiologic purposes can by achieved by the direct sequencing of a region with a moderate degree of genetic variability such as NS5B. HCV NS5B analysis is more efficient in resolving viral strain genetic variability than direct sequencing of a highly conserved region such as 5’UTR.

New Variant of Little cherry virus Associated with Little Cherry Disease of Sweet Cherry in British Columbia, Canada.

Little cherry disease (LChD) occurs in most cherry growing areas in the world. Infection of sensitive cultivars results in small fruit with poor color, angular shape, and insipid flavor. Three viruses associated with LCD have been described: (i) Little cherry virus-1 (LChV-1) first found and described in Germany (4); (ii) LChV-2 an isolate obtained from the United States (2); and (iii) LChV-3 first found and described in British Columbia (1). Despite similarities in symptom development in orchard trees and woody indexing, the three viruses have distinct molecular sequences (3). LChV-2 and -3 share greater homology with each other than either does with LChV-1. For many years, the British Columbia Ministry of Agriculture, Fisheries and Food (BCMAFF) in conjunction with Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre (AAFC-PARC) has conducted a survey to monitor the incidence and spread of LChD in the Okanagan and Kootenay valleys of British Columbia, Canada. Until recently, testing for LChD used woody indexing on indicator trees, Prunus avium cv. Lambert (fruit symptoms) and cvs. Canindex1 and Sam (foliar symptoms). Recently, incidence of LChD has been evaluated using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) developed at AAFC-PARC (3), and reverse transcription-polymerase chain reaction (RT-PCR) tests based on sequence data from LChV-3 (1). During the 1999 survey, orchard trees displaying symptoms typical of LChD tested negative for LChV-3 using ELISA and RT-PCR. Also, trees that formerly tested positive for LChD by woody indexing also tested negative for LChV-3 using RT-PCR and ELISA. Two hundred ninety-three trees were subsequently tested for LChV-1 by RT-PCR using the primer set LCV3EC/LCV16659 (4). A 276-bp fragment corresponding to the extreme 3' untranslated region (3' UTR) of the LChV-1 genome was amplified by RT-PCR from 140 of the trees. The RT-PCR amplicon from one sample (#99-68B from Peachland) was sequenced and using a BLASTn search, LChV-1 was identified as the most probable match (E value 4e-59). Sequence alignment using ClustalX identified two regions of high sequence homology; bases 2 to 70 (92%) and bases 99 to 239 (95%). The intervening region displayed much lower homology (61%). The overall homology of the amplicon was 88% compared to the corresponding region in LChV-1. Divergence between the published sequence of LChV-1 and the sequence of the new LChV isolate (tentatively named LChV-4) was investigated. Seven sets of primers constructed on the basis of sequence data from various regions of the LChV-1 genome (two sets from each of the RNA-dependent RNA polymerase, heat shock 70 protein homologue, and coat protein) failed to yield RT-PCR products when tested with the LChV-4 isolate. LChV-4 is clearly related to LChV-1 within the 3'-UTR but complete sequencing is required to determine the overall relationship with other viruses causing LChD. The discovery of a new isolate of LChV in British Columbia may require a reevaluation of the epidemiology of LChD and disease management strategy.

Extranuclear Estrogen-Regulated Destabilization ofXenopus laevisSerum Albumin mRNA

The present study examined 1) whether the estrogen-regulated destabilization of albumin mRNA occurs in the nuclear or extranuclear fraction of the liver cell, and 2) whether the selective posttranscriptional regulation of albumin mRNA stability might result from covalent changes introduced in the processing or polyadenylation of the primary transcript. The disappearance of albumin mRNA after estrogen is restricted to the extranuclear fraction of the cell. Transient changes in steady state levels of the mature nuclear transcript were observed that mirrored the transient estrogen-induced changes previously reported for albumin gene transcription. When assayed 24 h after estrogen (when albumin RNA is virtually undetectable in the extranuclear fraction) the steady state levels of both the primary and mature albumin transcripts found in the nucleus were the same as observed in control animals. Estrogen had no effect on the splicing or selection of polyadenylation sites on the 3'-UTR as determined by high resolution gel analysis of the 3'-UTR and DNA sequencing of cDNA clones isolated from a liver library from an estrogen-treated male Xenopus. Most eukaryotic mRNAs have poly(A) tracts several hundred residues in length, and recent studies have demonstrated that a change in the stability of a number of mRNAs correlates directly with the degree of polyadenylation. Albumin contrasts sharply with this, first because it has an exceptionally short poly(A) tail of 17 residues, and second because the degree of polyadenylation is totally unrelated to its destabilization in response to estrogen. These findings indicate that a unique pathway is involved in the regulation of albumin RNA stability by estrogen in Xenopus.