NTR Region Research Articles

Development and Validation of a One-Step Reverse Transcription Real-Time PCR Assay for Simultaneous Detection and Identification of Tomato Mottle Mosaic Virus and Tomato Brown Rugose Fruit Virus.

Tobamovirus species represent a threat to solanaceous crops worldwide, due to their extreme stability and because they are seed borne. In particular, recent outbreaks of tomato brown rugose fruit virus in tomato and pepper crops led to the establishment of prompt control measures, and the need for reliable diagnosis was urged. Another member of the genus, tomato mottle mosaic virus, has recently gained attention due to reports in different continents and its common features with tomato brown rugose fruit virus. In this study, a new real-time RT-PCR detection system was developed for tomato brown rugose fruit virus and tomato mottle mosaic virus on tomato leaves and seeds using TaqMan chemistry. This test was designed to detect tomato mottle mosaic virus by amplifying the movement protein gene in a duplex assay with the tomato brown rugose fruit virus target on the CP-3’NTR region, which was previously validated as a single assay. The performance of this test was evaluated, displaying analytical sensitivity 10−5–10−6-fold dilution for seeds and leaves, respectively, and good analytical specificity, repeatability, and reproducibility. Using the newly developed and validated test, tomato brown rugose fruit virus detection was 100% concordant with previously performed analyses on 106 official samples collected in 2021 from different continents.

Genetic characterisation of an Iflavirus associated with a vomiting disease in the Indian tropical tasar silkworm, Antheraea mylitta

Antheraea mylitta, the Tropical tasar silkworm, is frequently affected by a vomiting disease called Virosis by sericulturists although not confirmed being of viral origin. Based on the symptoms and the disease pattern, the causal agent is however suspected to be a virus. The condition involves a series of characteristic and progressive symptoms that generally culminates in the death of the larva. The disease is common in autumn season (Sep- Oct), with widespread distribution causing severe damage to the tasar silk industry. The leads for this study were obtained from a transcript identified in the EST database in a different study, which matched the positive strand of Iflavirus, an RNA virus known to infect insects. In the present study the genome of this novel Iflavirus was characterised and the full length of the genome was found to be 9728 nucleotides long encoding for a single large open reading frame (ORF) with flanking NTR regions at 5′ and 3′ ends and a natural poly A tail at the 3′ end. The ORF encoded structural proteins at the N-terminal end and non-structural proteins at the C-terminal end with a predicted 2967 amino acid long polyprotein. The structural proteins consisted of 4 proteins (VP1-VP4) and the non-structural proteins consisted of helicase, RNA-dependent RNA polymerase and 3C-protease. The virus is found in various tissues (midgut, fatbody, trachea, Malpighian tubules and silk gland) and also has a vertical route of transmission, i.e., from gravid females to the offspring. Based on the available data, the virus is a new member of Iflaviridae for which we propose the name Antheraea mylitta Iflavirus (AmIV).

Enterovirus D68 in a community hospital: A test-negative case-control study

Background: In August 2014, a large North American outbreak of enterovirus D68 (EV-D68) was observed in association with severe respiratory illnesses in children as well as a possible association with acute flaccid paralysis. The full spectrum of clinical illness associated with EV-D68 is not well known. Objective: To compare children positive and negative for EV-D68 presenting to a community hospital during the North American outbreak in the fall of 2014. Methods: Consecutive nasopharyngeal swabs obtained from children (<18 years old) presenting to a large community hospital with respiratory symptoms in the fall of 2014 were tested for EV-D68. We conducted a test-negative case-control study between EV-D68 positive and EV-D68 negative children to describe the clinical and outcome characteristics. Controls were limited to children who had a nasopharyngeal swab obtained. EV-D68 indeterminate cases were excluded from the primary analysis. All nasopharyngeal swabs were referred to a reference laboratory for polymerase chain reaction testing and identification of EV-D68 through targeting the 5'NTR region and confirmed by amplifying the VP1 gene for serotype determination. Results: Of the 48 children tested there were 9 positive, 36 negative, and 3 indeterminate for EV-D68. Six of the 9 identified cases of EV-D68 were identified in the first week of testing (September 11–18, 2014). There were 35 children (78%) admitted to hospital for ≥1 day. EV-D68 infected children were significantly older (5.2 versus 2.5 years, p = 0.03). They were more likely to be concurrently taking asthma medications (44% versus 11%, p = 0.04) and receiving treatment with salbutamol (100% versus 58%, p = 0.02). There were no cases of severe respiratory distress or acute flaccid paralysis. Three EV-D68 infected children (33%) had viral co-infections identified. Conclusion: This study of a community hospital experience during the fall of 2014 demonstrates a series of mild illnesses from both EV-D68 positive and negative children. EV-D68 isolation was associated with age and asthmatic treatment in children.

Development of a PCR protocol for the detection of human enteroviruses based on the analysis of the 5'-NTR region and its use in molecular epidemiology

Development of a PCR protocol for the detection of human enteroviruses based on the analysis of the 5'-NTR region and its use in molecular epidemiology

Characterization of significant molecular determinants of virulence of Enterovirus 71 sub-genotype B4 in Rhabdomyosarcoma cells

One of the leading causes of the hand, foot and mouth disease (HFMD) is Enterovirus 71 (EV-A71), displaying symptoms such as fever and ulcers in children but some strains can produce cardiopulmonary oedema which leads to death. There is no FDA-approved vaccine for prevention of severe HFMD. The molecular determinants of virulence for EV-A71 are unclear. It could be a single or a combination of amino acids that determines virulence in different EV-A71 genotype/sub-genotypes. Several EV-A71 strains bearing single nucleotide (nt) mutations were constructed and the contribution of each mutation to virulence was evaluated. The nt(s) that contributed to significant reduction in virulence in vitro were selected and each mutation was introduced separately into the genome to construct the multiply mutated EV-A71 strain (MMS) which carried six substitutions of nt(s) at the 5′-NTR (U700C), VP1-145 (E to G), VP1-98E, VP1-244K and G64R in the vaccine seed strain that had a partial deletion within the 5′-NTR region (nt. 475–485) of Δ11bp. In comparison to the wild type strain, the MMS showed low virulence as it produced very low RNA copy number, plaque count, VP1 and had 105-fold higher TCID50, indicative of a promising LAV candidate that should be further evaluated in vivo.

Identification of molecular determinants of cell culture growth characteristics of Enterovirus 71.

BackgroundHand, foot and mouth disease is caused by Enterovirus 71 (EV-A71) and Coxsackieviruses. EV-A71 infection is associated with high fever, rashes and ulcers but more severe symptoms such as cardiopulmonary failure and death have been reported. The lack of vaccines highlighted the urgency of developing preventive agents against EV-A71. The molecular determinants of virulent phenotypes of EV-A71 is unclear. It remains to be investigated if specific molecular determinants would affect the cell culture growth characteristics of the EV-A71 fatal strain in Rhabdomyosarcoma (RD) cells.ResultsIn this study, several genetically modified sub-genotype B4 EV-A71 mutants were constructed by site-directed mutations at positions 158, 475, 486, 487 and 5262 or through partial deletion of the 5′-NTR region (∆ 11 bp from nt 475 to 486) to generate a deletion mutant (PD). EV-A71 mutants 475 and PD caused minimal cytopathic effects, produced lowest viral RNA copy number, viral particles as well as minimal amount of viral protein (VP1) in RD cells when compared to mutants 158, 486, 487 and 5262.ConclusionsThe molecular determinants of virulent phenotypes of EV-A71 sub-genotype B4 strain 41 (5865/Sin/000009) were found to differ from the C158 molecular determinant reported for the fatal EV-A71 sub-genotype B1 strain (clinical isolate 237). The site-directed mutations (SDM) introduced at various sites of the cDNA affected growth of the various mutants when compared to the wild type. Lowest viral RNA copy number, minimal number of plaques formed, higher infectious doses required for 50% lethality of RD cells and much reduced VP1 of the EV-A71 sub-genotype B4 strain 41 genome was attained in mutants carrying SDM at position 475 and through partial deletion of 11 bp at the 5′-NTR region.

Effects of Hfq on the Conformation and Compaction of DNA

Hfq is a bacterial pleiotropic regulator that mediates several aspects of nucleic acids metabolism. The protein notably influences translation and turnover of cellular RNAs. Although most previous contributions concentrated on Hfq's interaction with RNA, its association to DNA has also been observed in vitro and in vivo. Here, we focus on DNA-compacting properties of the Hfq hexamer as well as the NTR and CTR regions. Various experimental technologies, including fluorescence microscopy imaging of single DNA molecules confined inside nanofluidic channels, atomic force microscopy and small angle neutron scattering have been used to follow the assembly of Hfq on DNA. Our results show that Hfq forms a nucleoprotein complex, changes the mechanical properties of the double helix and compacts DNA into a condensed form. We propose a compaction mechanism based on protein-mediated bridging of DNA segments. The propensity for bridging is related to multi-arm functionality of the Hfq hexamer, resulting from binding of the C-terminal domains to the duplex.

Isolation and molecular characterization of bovine enteroviruses in Egypt

Enteroviruses belong to the Picornaviridae family and infect a wide range of mammals including cattle. Bovine enterovirus (BEV) has recently been reclassified into E and F serotypes. BEV was first isolated in Egypt in 1966 although it has been known in other countries since the 1950s. In this study, BEV-F2 was isolated from calves with severe diarrhea and the isolated viruses were subjected to molecular characterization. Illumina sequencing of one of the isolates revealed the presence of a complete BEV-F genome sequence. The phylogenetic analysis revealed nucleotide substitutions along the genome in comparison with other known strains of BEV-F (HQ663846, AY508697 and DQ092795). Two primer sets were designed from the 3D and 5′NTR regions and used for the examination of the remaining isolates, which were confirmed to be of the BEV-F2 serotype. The availability of the complete genome sequence of this virus adds to the sequence database of the members of Picornaviridae and should be useful in future molecular studies of BEV.

Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India. Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains. A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable. Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

Validation of a real time PCR for classical Swine Fever diagnosis.

The viral disease classical swine fever (CSF), caused by a Pestivirus, is one of the major causes of economic losses for pig farming. The aim of this work was to validate a RT-qPCR using Taqman for detection of CSF in swine tissues. The parameters for the validation followed the specifications of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the World Organization for Animal Health (OIE) and the guide ABNT NBR ISO/IEC 17025:2005. The analysis of the 5′NTR region of CSF virus was performed in 145 samples from 29 infected pigs and in 240 samples from 80 pigs originated in the Brazilian CSF-free zone. The tissues tested were spleen, kidney, blood, tonsils, and lymph nodes. Sequencing of the positive samples for 5′NTR region was performed to evaluate the specificity of the RT-qPCR. Tests performed for the RT-qPCR validation demonstrated that the PCR assay was efficient in detecting RNA from CSF virus in all materials from different tissues of infected animals. Furthermore, RNA from CSF virus was not detected in samples of swine originated from the Brazilian CSF-free zone. Hence, it is concluded that RT-qPCR can be used as a complementary diagnostic for CSF.

Evaluation of Hepatitis C Virus Genotyping Protocols for Use in a Diagnostic Setting

Background: Hepatitis C virus (HCV) has been reported to be the most common cause of chronic viral Hepatitis with increased prevalence rate worldwide. HCV genotype is an important predictive factor in the outcome of HCV treatment, therefore an essential component of clinical practice. Molecular methods are the most common diagnostic tools for HCV genotyping. HCV genotype might also have relevance for newer antiviral therapies in development. Objectives: To develop and evaluate new cost effective molecular methods for HCV genotyping using analytical instruments which were in place at Royal Liverpool University Hospital (RLUH) and to assess their sensitivity and specificity. Methods: The plasma or serum RNA purification was done by automated QIAsymphony and QIAcube equipment. PCR assays capable of detecting and distinguishing HCV genotype by nucleotide probe or melt analysis were compared. The two-step PCR and one-step real-time PCR methods were assessed. The NS5B PCR was used to amplify the 5'NTR region of HCV for sequencing using Qiagen RT-PCT kit. The method was evaluated using HCV genomic sequence data as the gold standard. Results: In preliminary PCR assessments, two-step real-time PCR amplified the products but could not be continued as efforts were made to assess and improve the one-step RT-PCR method for diagnostic purposes. Out a total of 71 samples 65 gave definitive genotype results, 59 were concordant and 6 mismatched. The discordant samples were repeated to confirm the genotypes but the results were similar. 6 samples could not be amplified. A Kappa value of 0.869 (p<0.001) indicating a 90.8 percent agreement was obtained. The sensitivity and specificity were 91.5% and 100% respectively. Conclusions: The PCR amplification of NS5B HCV regions showed that the method is capable of detecting HCV genotypes, however it is unable to discriminate mixed genotypes. Whereas real-time PCR method is capable of detecting mixed genotypes, the genotype call may be less reliable. On the other hand, the sequencing method used in this study has an inherent limitation of giving definitive genotypes one at a time. Further efforts should be made to develop the real-time PCR to detect mixed genotypes.

Potato virus surveys and wide spread of recombinant PVYNTN variant in Central Tunisia

Surveys in the late season crops of Central Tunisia, one of the main potato growing areas, revealed the presence of the six most economically devastating viruses: potato leaf roll virus: PLRV: (Polerovirus), potato virus S (PVS) and M (PVM) (Carlavirus), potato virus X (PVX: Potexvirus), potato virus A (PVA) and Y (PVY) (Potyvirus) with large differences in incidence. Infection rates at harvest, assessed by serological test ranged from 0.5% (PVM) to 71% (PVY). The characterization of PVY variability was analyzed with a combination of serotyping, indexing on tobacco and reverse transcription polymerase chain reaction (RT-PCR) tests targeting 2 genomic regions (5’NTR/P1 and CP/3’NTR). Serological analysis of the collected samples revealed dominance of the PVYN group (88.2% of the total number of singly PVY positives). Furthermore, molecular typing of strains revealed that 73.3% of the PVYN group were typical PVYNTN variants with a recombination junction in CP/3’NTR region for 94.4% of them, whereas no recombination junction was identified in the genome of the isolates belonging to the PVYN group. To our knowledge, this is the first report of the occurrence of the PVYNTN variant and its high incidence in late season potato growing areas of central Tunisia. Key words: Diagnosis, immunocapture reverse transcription polymerase chain reaction (RT PCR), potato viruses, potato virus Y (PVY) diversity, Central Tunisia.

Molecular Analysis of Virulent Determinants of Enterovirus 71

Enterovirus 71 (EV71) is the most important causative agent of hand, foot and mouth disease (HFMD) in children. In most cases, it is a self-limiting illness. However some EV71 infectious cases can develop severe clinical outcomes, such as encephalitis, meningitis, poliomyelitis like paralysis, and even death. To identify the determinants of virulence, the deduced amino acid sequence of polyprotein and nucleotide sequence of 5′-NTR and 3′-NTR in 25 SC-EV71 strains (strains from severe cases) and 31 MC-EV71 strains (strains from mild cases) were analyzed. Results showed four amino acids on two positions (GlyP710/GlnP710/ArgP710 and GluP729) on the DE and EF loop of VP1, one (LysP930) on the surface of protease 2A and four nucleotides on three positions (GP272, UP488 and AP700/UP700) in the 5'-NTR region are associated with EV71 virulent phenotype. Predicted secondary structure of RNA using the consensus sequence of 5'-NTR by RNAStructure showed the mutation of nucleotide at position 488 in strain BJ08-Z004-3 (position 491 in prototype strain BrCr) can result in the discrepancy of an additional pair of nucleotides and thus change the stability of the second structure of IRES. Fragment base content analysis showed that in the region 696 to 714 bp at the 5'-NTR, where the AP700/UP700 was located, the nucleotide constitution ratios differed significantly between SC-EV71 and MC-EV71 strains. In conclusion, comparative genomic analysis showed that virulence of EV71 strains are mainly determined by the amino acids on two positions of VP1, one position of protease 2A and the nucleotides on three positions in 5'-NTR.

Aseptic meningitis by echovirus 30 in São Paulo state, Brazil

The objective of this study was to identify and characterize the immunological and molecular aspects of Human Enterovirus (HEV) associated with aseptic meningitis cases in Sao Paulo State, Brazil. Clinical samples (cerebrospinal fluid CSF, stool and sera) were analyzed from 21 viral meningitis suspected cases collected in the period from 1998 to 1999. The isolated virus were identified as belonging to Enterovirus genus and specifically to echovirus serotype 30 (EV-30), using immunological techniques as Indirect Immunofluorescense Assay (IFA) and Neutralization (Nt) and by molecular technique of Reverse Transcription Polymerase Chain Reaction (RT-PCR) directed to the 5' NTR and VP1 regions of viral genome. The results obtained contributed with to the surveillance system of Sao Paulo State and they are important due to a shortage of epidemiological data about the circulation of EV-30 associated to aseptic meningitis in this region of the world.

Co-circulation of genotypes IA and IB of hepatitis A virus in Northeast Brazil

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Interaction of glyceraldehyde-3-phosphate dehydrogenase with secondary and tertiary RNA structural elements of the hepatitis A virus 3' translated and non-translated regions.

Proteins interacting with RNA structures at the 3' non-translated region (3'NTR) of picornaviruses are probably important during viral RNA replication. We have shown previously that a dominant cellular cytoplasmic protein of 38 kDa (p38) interacts with the 3'NTR and upstream regions of the hepatitis A virus (HAV) RNA (Kusov et al., J Virol 70, 1890-1897, 1996). Immunological and biochemical analyses of p38 have indicated that it is identical to GAPDH, which has previously been described as modulating translational regulation of the HAV RNA by interacting with the 5'NTR (Schultz et al., J Biol Chem 271, 14134-14142, 1996). Three separate binding regions for GAPDH in the 3'NTR and in the upstream 3D polymerase-coding region were identified. Structural analysis of these RNA regions by computer modelling and direct enzymatic cleavage suggested the presence of several AU-rich stem-loop structures having the potential for tertiary interactions. Binding of GAPDH to these structures was confirmed by RNA footprint analysis and resulted in the loss of double-stranded RNA regions. A different panel of RNA binding proteins (p28, p41 and p65) was detected in the ribosomal fractions of several cell lines (BSC-1, FRhK-4 and HeLa), whereas RNA binding of the GAPDH that was also present in these fractions was only marginal or absent.

Virolytic action of lipoprotein lipase on hepatitis C virus in human sera.

In most sera of hepatitis C virus (HCV)-infected patients beta-lipoproteins are bound to HCV RNA-carrying material, most often simultaneously with immunoglobulins (IgG, IgM) and sometimes additionally with high-density lipoproteins, forming complexes of low density (1.04-1.06 g/ml). To separate HCV particles from bound material, we tried to destroy the lipoprotein enzymatically by incubating HCV-positive human sera with lipoprotein lipase derived from Pseudomonas spp. (LPL-Ps). After this treatment, titers of HCV RNA in human sera (determined by a simple semiquantitative reverse transcription-PCR assay) were strongly reduced, regardless of whether primers of the NTR or NS5 region were used. Inactivation of HCV RNA could be inhibited by the addition of RNAguard to the serum-enzyme mixture. The lytic effect of the LPL-Ps preparation could be inhibited by tetrahydrolipstatin. Hence LPL-Ps seems to disrupt the HCV particle structure, including the putative core of the virus, and makes HCV RNA sensitive for RNase present in the reaction mixture. HBV was not destroyed by LPL-Ps. Porcine pancreatic lipase had no effect on HCV. The implications of these observations for the structure and biology of HCV and for its stability and inactivation in human sera are discussed.

Molecular Characterization of Potato virus V Genomes from Europe Indicates Limited Spatiotemporal Strain Differentiation

ABSTRACT Because there were no previous reports on the molecular characterization of Potato virus V (PVV, genus Potyvirus, family Potyviridae), the complete genomic sequence of PVV isolate Dv42 was determined. The length of the single-stranded messenger-polarity RNA genome was 9,851 nt (nucleotides), followed by a poly(A) tail. The genome contained a 5'-terminal nontranslated region (5'-NTR; 204 nt), a single open reading frame (nucleotides 205-9406; 3,067 amino acids), and a 3'-NTR that was unusually long (446 nt) compared with that of Potato virus Y (PVY; 331-nt 3'-NTR), Potato virus A (PVA; 207-nt 3'-NTR), and other potyviruses that naturally infect Solanaceae species. Phylogenetic analysis with the cylindrical inclusion protein-encoding and coat protein (CP)-encoding regions indicated that PVV Dv42 was most closely related to Pepper mottle virus and PVY, respectively. Seven PVV isolates (including Dv42) collected from cultivated potatoes in the Netherlands, the United Kingdom, and Norway from 1964 to 1997 were uniform in serological properties and symptomatology in indicator hosts that could distinguish strains of PVY and PVA. The nucleotide sequences of the 5'-NTR, P1, CP, and 3'-NTR regions of the PVV isolates were determined and were 94.6 to 99.5, 96.3 to 98.8, 96.4 to 98.7, and 96.3 to 99.6% identical, respectively. The amino acid similarities for the P1 and CP were 95.8 to 98.6 and 96.0 to 97.8%, respectively. Phylogenetic analysis of the CP sequences of PVV revealed no significant grouping, in contrast to PVY and PVA, which were grouped largely according to the previously recognized strains based on host responses. However, the relatively few differences in the P1 sequences of PVV were correlated with the different countries of origin. Hence, the PVV isolates infecting potatoes in Europe seem to vary little genetically and may belong to a single strain.