RNA Reverse Transcription PCR Research Articles

Detectable Duration of Viable SARS-CoV-2, Total and Subgenomic SARS-CoV-2 RNA in Noncritically Ill COVID-19 Patients: a Prospective Cohort Study.

ABSTRACTDetermination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is important in guiding the infection control and differentiating between reinfection and persistent viral RNA. Although viral culture is the gold standard to determine viral infectivity, the method is not practical. We studied the kinetics of SARS-CoV-2 total RNAs and subgenomic RNAs (sgRNAs) and their potential role as surrogate markers of viral infectivity. The kinetics of SARS-CoV-2 sgRNAs compared to those of the culture and total RNA shedding in a prospective cohort of patients diagnosed with coronavirus disease 2019 (COVID-19) were investigated. A total of 260 nasopharyngeal swabs from 36 patients were collected every other day after entering the study until the day of viral total RNA clearance, as measured by reverse transcription PCR (RT-PCR). Time to cessation of viral shedding was in order from shortest to longest: by viral culture, sgRNA RT-PCR, and total RNA RT-PCR. The median time (interquartile range) to negativity of viral culture, subgenomic N transcript, and N gene were 7 (5 to 9), 11 (9 to 16), and 18 (13 to 21) days, respectively (P < 0.001). Further analysis identified the receipt of steroid as the factors associated with longer duration of viral infectivity (hazard ratio, 3.28; 95% confidence interval, 1.02 to 10.61; P = 0.047). We propose the potential role of the detection of SARS-CoV-2 subgenomic RNA as the surrogate marker of viral infectivity. Patients with negative subgenomic N RNA RT-PCR could be considered for ending isolation.IMPORTANCE Our study, combined with existing evidence, suggests the feasibility of the use of subgenomic RNA RT-PCR as a surrogate marker for SARS-CoV-2 infectivity. The kinetics of SARS-CoV-2 subgenomic RNA should be further investigated in immunocompromised patients.

A novel spliced gene in alcelaphine herpesvirus 1 encodes a glycoprotein which is secreted in vitro

Herpesviruses often contain cryptic, spliced genes that are not obvious from the initial in silico annotation. Alcelaphine herpesvirus 1 (AlHV-1) contains 72 annotated ORFs but there are also a number of gaps between these that may have protein-coding potential. Comparative analysis of coding potential between AlHV-1 and the related ovine herpesvirus 2 (OvHV-2) revealed a putative novel spliced gene that we have termed A9.5. Analysis of cDNA clones from AlHV-1-infected cells revealed three overlapping clones corresponding to A9.5 and the coding sequence was confirmed by reverse transcription PCR of RNA from AlHV-1-infected cattle tissues. The A9.5 gene was predicted to encode a secreted glycoprotein with molecular mass 19 kDa. Empirical analysis showed that a recombinant haemagglutinin-tagged A9.5 fusion protein was secreted from transfected cells and had a molecular mass of 45 kDa, which was reduced to 20 kDa by endoglycosidase F treatment, confirming that A9.5 was a secreted glycoprotein. In situ RNA hybridization showed that A9.5 was expressed in cells associated with malignant catarrhal fever (MCF) lesions in infected cattle. Detailed analysis of the available OvHV-2 sequences revealed an homologous gene (Ov9.5) with conserved splicing signals and predicted amino acid sequence features in both sequenced isolates of this related virus. We have therefore identified a novel spliced gene in two related macaviruses that is expressed in MCF lesions. Future work will determine its importance for the pathogenesis of disease.

West Nile Virus Infection in Belgian Traveler Returning from Greece

West Nile Virus Infection in Belgian Traveler Returning from Greece

Transmission of hepatitis C virus infection from asymptomatic mother to child in southern India

Little information is available on the mother-to-child transmission of hepatitis C virus (HCV) in India, and no interventions to decrease transmission rates have been identified. Hence, we performed a long-term prospective study in infants born to HCV-positive mothers, with the aim of evaluating vertical transmission of HCV and correlated risks factors. Three thousand one hundred and fifteen healthy asymptomatic pregnant women were included in the study. We used third-generation (Murex anti-HCV) ELISA and HCV RNA reverse transcription PCR (RT-PCR) for screening, and the commercial line probe assay (Inno-LiPA) and direct sequencing HCV genotyping assays were performed to confirm the transmitted HCV genotypes. Of the total 3115 healthy asymptomatic pregnant women, 18 (0.6%) were positive for anti-HCV. Of the 18 anti-HCV-positive women, eight (44.4%) were positive for HCV RNA RT-PCR. HCV transmission was observed in two of the eight babies born to eight HCV RNA-positive mothers who were followed up for 12 months. HCV genotyping of the mother/child pairs revealed the persistent presence of mixed genotypes 1a and 4 throughout the follow-up period. None of the non-viremic (HCV RNA-negative) mothers transmitted HCV infection to their baby. In our study approximately 25% of vertical/perinatal transmission of HCV was observed among HCV RNA-positive antenatal women. This study is of importance as it is the first report from India of a successful attempt to analyze the rate of vertical/perinatal transmission of HCV from infected mothers to their children by a prospective longitudinal follow-up study, and to characterize the pattern of genotype(s) of HCV present in the infected mother/baby pairs, so as to confirm the source of HCV acquired by the newborn babies.

Candidate New Species ofKobuvirusin Porcine Hosts

Candidate New Species of<i>Kobuvirus</i>in Porcine Hosts

Expression of β-galactosidase and β-xylosidase genes during microspore and pollen development

Tobacco (Nicotiana tabacum L.) microspores at the time of mitosis are characterized by the abundant occurrence of 92- and 98-kDa glycoproteins (GP92 and GP98). GP92 is a soluble protein while GP98 is bound to the insoluble microspore fraction. Both glycoproteins were isolated by affinity chromatography and SDS-PAGE and analysed by MS. Peptide sequences were determined by mu-HPLC/nano-ESI-MS/MS (electrospray ionization tandem MS). GP92 displayed homology to beta-galactosidase (EC 3.2.1.23) and GP98 to beta-xylosidase (EC 3.2.1.37) from Arabidopsis thaliana (L.) Heynh. The activities of the two enzymes in microspore and pollen extracts of tobacco exhibited similar developmental changes to the occurrence of GP92 and GP98, with a maximum around microspore mitosis. These two glycoproteins are the first identified enzymes characteristic of mitotic microspores. Arabidopsis transcriptomic data for five beta-galactosidase and three beta-xylosidase genes abundantly expressed in pollen were verified by reverse transcription-PCR of RNA from different stages of Arabidopsis pollen development and from various parts of the sporophyte. The results showed abundant expression of two genes (At5g20710, At1g31740) homologous to tobacco GP92 in microspores and early pollen, and of three genes (At5g56870, At2g16730 and At4g35010) in maturing pollen. Analysis of beta-xylosidases showed abundant expression of a late pollen-specific gene At3g62710 and low expression of an early gene At5g10560. It is suggested that the early beta-galactosidase and beta-xylosidase genes may participate in cell wall loosening associated with pollen expansion after microspore mitosis and that the products of the late genes may play a role in cell expansion during pollen germination.

Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively.

Molecular phylogenetic analyses of reverse-transcribed bacterial rRNA obtained from deep-sea cold seep sediments.

A depth profile of naturally occurring bacterial community structures associated with the deep-sea cold seep push-core sediment in the Japan Trench at a depth of 5343 m were evaluated using molecular phylogenetic analyses of RNA reverse transcription-PCR (RT-PCR) amplified 16S crDNA fragments. A total of 137 clones of bacterial crDNA (complimentary rDNA) phylotypes (phylogenetic types) obtained at three different depths (2-4, 8-10 and 14-16 cm) were identified in partial crDNA sequencings. crDNA phylotypes from the cold seep sediment were dominantly composed of delta- and epsilon-Proteobacteria (36% and 42% respectively). Phylotype analysis of crDNA clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the majority of bacterial components shifted from delta- Proteobacteria to epsilon-Proteobacteria with increasing depth. Among the delta-proteobacterial crDNA clones, the sequences related to the genus Desulfosarcina were dominant. Almost all sequences of crDNA belonging to epsilon-Proteobacteria were affiliated with the same cluster (epsilon-CSG: epsilon-proteobacterial cold seep group), and were closely related with rDNA sequences from deep-sea hydrothermal vent environments.

The expression of renin and the formation of angiotensin II in bovine aortic endothelial cells.

One controversy in the field of vascular angiotensin generation has surrounded the nature and particularly the source of vascular renin. This study investigated the expression of renin protein and its mRNA in aortic endothelial cells using immunocytochemistry, Western blotting, in situ hybridization and reverse transcription PCR (RT-PCR). Using a monoclonal antibody against human renin, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Immunoblotting of solubilized proteins separated by SDS-PAGE from cultured aortic endothelial cells identified two immunoreactive species with molecular masses of approximately 37-40 kDa. In situ hybridization showed that renin mRNA was localized in the cytoplasm of these cells. Using RT-PCR of RNA extracted from bovine aortic endothelial cells with primers specific for human renin, a clear single band was detected, which had the predicted size of 142 bp for (pro)renin. Angiotensin II (Ang II) was assayed in conditioned medium (CM) from cultured bovine aortic endothelial cells, and in addition, the effects of Ang II and CM on the proliferation of aorta smooth muscle cells (ASMC) were also studied. The results showed that CM contained Ang II equivalent to 15.05+/-4.67 pg/10(6) cells. Assay of smooth muscle cell proliferation by cell number, and by tritiated thymidine uptake, showed that proliferative responses in the presence of Ang II at a concentration of 10(-6)M were evident within 1 day of subculture, and cell numbers were nearly twice those of controls after 2 days. Thymidine incorporation into ASMC was also increased by Ang II in a dose-dependent manner and by endothelial cell CM. In both cases, stimulated proliferation was inhibited by the Ang II type 1 (AT1) receptor selective antagonist, losartan. These findings suggest that these vascular endothelial cells are a source of locally synthesized renin that may thus be involved in vascular Ang II generation. They also suggest that Ang II produced by the endothelial cells may be secreted and stimulate ASMC proliferation via the AT1 receptor.

Reverse transcription-PCR analysis of the regulation of the manganese peroxidase gene family.

Manganese peroxidase (MnP) gene expression in the lignin-degrading fungus Phanerochaete chrysosporium is regulated by nutrient nitrogen levels and by Mn(II), the substrate for the enzyme, as well as by heat shock and other factors. Reverse transcription-PCR (RT-PCR) of total RNA can distinguish the mRNAs of each of the three sequenced P. chrysosporium mnp genes, i.e., mnp1, mnp2, and mnp3. Quantitative RT-PCR demonstrates that each of the three transcripts is present at a similar low basal level in nitrogen-sufficient cultures, with or without Mn, and in nitrogen-limited cultures lacking Mn. However, in 5-day-old, nitrogen-limited, stationary cultures supplemented with 180 microM Mn, the levels of the mnp1 and mnp2 transcripts increased approximately 100- and 1,700-fold, respectively, over basal levels. In contrast, under these conditions, the level of the mnp3 transcript did not increase significantly over the basal level. Quantitative RT-PCR of total RNA extracted from nitrogen-deficient, Mn-supplemented cultures on days 2 through 7 demonstrates that whereas the mnp1 transcript was present at relatively low levels on days 3 through 7, the mnp2 transcript level peaked on day 5 and the mnp3 transcript level peaked on day 3. Comparison of total RNA extracted on day 5 from nitrogen-deficient, Mn-supplemented stationary and agitated cultures indicates that in stationary cultures, mnp2 was the major expressed mnp gene, whereas in large agitated cultures, mnp1 was the major expressed mnp gene.

High-throughput RT-PCR analysis of multiple transcripts using a microplate RNA isolation procedure.

We have developed a high-throughput, multiplex reverse transcription PCR (RTPCR) assay that is suitable for the analysis of medium-to low-copy cellular RNA transcripts from small numbers of cells (10(4)). High throughput was attained by utilizing microplate-based RNA extraction and RTPCR protocols, followed by PCR product visualization of a multiwelled agarose gel, stained with SYBR Green I dye. The transcriptional assay was unaffected by solvents (dimethyl sulfoxide and methanol) routinely used in high-throughput drug screens at concentrations required for compound solubilization. Furthermore, it has been used successfully for the investigation of differential mRNA expression levels of tumor necrosis factor alpha (TNF-alpha) and Interleukin-1 beta (IL-1 beta) in lipopolysaccharide (LPS)-stimulated THP-1 cells (a human monocytic cell line) and the identification of specific IL-1 beta transcriptional inhibitors.

Detection of hepatitis A virus RNA in oyster meat.

Detection of low concentrations of viruses in shellfish is possible with nucleic acid amplification by PCR. Hepatitis A virus (HAV) has been detected in oyster meat by reverse transcription-PCR (RT-PCR). We developed a method to identify HAV RNA by RT-PCR of total RNA extracted from oyster meat contaminated by adsorption, bioaccumulation, or injection. With dot blot hybridization detection of amplicons from the RT-PCR, rapid screening of a large number of samples is feasible. As few as 8 PFU of HAV/g of oyster meat can be detected.