Identification Of Viral Nucleic Acid Research Articles

Usefulness of ELISA Using Total Antibody against Plant-Expressed Recombinant Nucleocapsid Protein of SARS-CoV-2.

ABSTRACTHere, we aimed to investigate the diagnostic value of a serological assay using the nucleocapsid protein developed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection and evaluated its performance using three commercial enzyme-linked immunosorbent assays (ELISAs), namely, Standard E 2019 novel coronavirus disease (COVID-19) total antibody (Ab) ELISA (SD Biosensor), and EDI novel coronavirus COVID-19 IgG and IgM ELISA. A recombinant nucleocapsid protein (rNP) was expressed from plants and Escherichia coli for the detection of serum total Ab. We prospectively collected 141 serum samples from 32 patients with reverse transcription-PCR (RT-PCR)-confirmed COVID-19 and determined the sensitivity and dynamics of their total Ab response. Specificity was evaluated using 158 prepandemic samples. To validate the assays, we evaluated the performance using two different cutoff values. The sensitivity and specificity for each assay were as follows: 92.91% and 94.30% (plant-rNP), 83.69% and 98.73% (SD Biosensor), 75.89% and 98.10% (E. coli-rNP), 76.47% and 100% (EDI-IgG), and 80.39% and 80% (EDI-IgM). The plant-based rNP showed the highest sensitivity and area under the receiver operating characteristic (ROC) curve (0.980) among all the assays (P < 0.05). The seroconversion rate for total Ab increased sequentially with disease progression, with a sensitivity of 100% after 10 to 12 days of post-symptom onset (PSO) for both rNP-plant-based and SD Biosensor ELISAs. After 2 weeks of PSO, the seroconversion rates were >80% and 100% for EDI-IgM and EDI-IgG ELISA, respectively. Seroconversion occurred earlier with rNP plant-based ELISA (5 days PSO) compared with E. coli-based (7 days PSO) and SD Biosensor (8 days PSO) ELISA. We determined that rNP produced in plants enables the robust detection of SARS-CoV-2 total Abs. The assay can be used for serosurvey and complementary diagnosis of COVID-19.IMPORTANCE At present, the principal diagnostic methods for COVID-19 comprise the identification of viral nucleic acid by genetic approaches, including PCR-based techniques or next-generation sequencing. However, there is an urgent need for validated serological assays which are crucial for the understanding of immune responses against SARS-CoV-2. In this study, a highly sensitive and specific serological antibody assay was developed for the detection of SARS-CoV-2 with an overall accuracy of 93.56% using a recombinant nucleoprotein expressed from plants.

Pattern of Respiratory Viruses among Pilgrims during 2019 Hajj Season Who Sought Healthcare Due to Severe Respiratory Symptoms.

The aim of our study was to define the spectrum of viral infections in pilgrims with acute respiratory tract illnesses presenting to healthcare facilities around the holy places in Makkah, Saudi Arabia during the 2019 Hajj pilgrimage. During the five days of Hajj, a total of 185 pilgrims were enrolled in the study. Nasopharyngeal swabs (NPSs) of 126/185 patients (68.11%) tested positive for one or more respiratory viruses by PCR. Among the 126 pilgrims whose NPS were PCR positive: (a) there were 93/126 (74%) with a single virus infection, (b) 33/126 (26%) with coinfection with more than one virus (up to four viruses): of these, 25/33 cases had coinfection with two viruses; 6/33 were infected with three viruses, while the remaining 2/33 patients had infection with four viruses. Human rhinovirus (HRV) was the most common detected viruses with 53 cases (42.06%), followed by 27 (21.43%) cases of influenza A (H1N1), and 23 (18.25%) cases of influenza A other than H1N1. Twenty-five cases of CoV-229E (19.84%) were detected more than other coronavirus members (5 CoV-OC43 (3.97%), 4 CoV-HKU1 (3.17%), and 1 CoV-NL63 (0.79%)). PIV-3 was detected in 8 cases (6.35%). A single case (0.79%) of PIV-1 and PIV-4 were found. HMPV represented 5 (3.97%), RSV and influenza B 4 (3.17%) for each, and Parechovirus 1 (0.79%). Enterovirus, Bocavirus, and M. pneumoniae were not detected. Whether identification of viral nucleic acid represents nasopharyngeal carriage or specific causal etiology of RTI remains to be defined. Large controlled cohort studies (pre-Hajj, during Hajj, and post-Hajj) are required to define the carriage rates and the specific etiology and causal roles of specific individual viruses or combination of viruses in the pathogenesis of respiratory tract infections in pilgrims participating in the annual Hajj. Studies of the specific microbial etiology of respiratory track infections (RTIs) at mass gathering religious events remain a priority, especially in light of the novel SARS-CoV-2 pandemic.

Rapid Serological Assays and SARS-CoV-2 Real-Time Polymerase Chain Reaction Assays for the Detection of SARS-CoV-2: Comparative Study

BackgroundReal-time polymerase chain reaction (RT-PCR) testing for the identification of viral nucleic acid is the current standard for the diagnosis of SARS-CoV-2 infection, but technical issues limit its utilization for large-scale screening. Serological immunoglobulin M (IgM)/IgG testing has been proposed as a useful tool for detecting SARS-CoV-2 exposure.ObjectiveThe objective of our study was to compare the results of the rapid serological VivaDiag test for SARS-CoV-2–related IgM/IgG detection with those of the standard RT-PCR laboratory test for identifying SARS-CoV-2 nucleic acid.MethodsWe simultaneously performed both serological and molecular tests with a consecutive series of 191 symptomatic patients. The results provided by a new rapid serological colorimetric test for analyzing IgM/IgG expression were compared with those of RT-PCR testing for SARS-CoV-2 detection.ResultsOf the 191 subjects, 70 (36.6%) tested positive for SARS-CoV-2 based on RT-PCR results, while 34 (17.3%) tested positive based on serological IgM/IgG expression. Additionally, 13 (6.8%) subjects tested positive based on serological test results, but also tested negative based on RT-PCR results. The rapid serological test had a sensitivity of 30% and a specificity of 89% compared to the standard RT-PCR assay. Interestingly, the performance of both assays improved 8 days after symptom appearance. After 10 days had passed since symptom appearance, the predictive value of the rapid serological test was higher than that of the standard molecular assay (proportion of positive results: 40% vs 20%). Multivariate analysis showed that age >58 years (P<.01) and period of >15 days after symptom onset (P<.02) were significant and independent factors associated with serological test positivity.ConclusionsThe rapid serological test analyzed in this study seems limited in terms of usefulness when diagnosing SARS-CoV-2 infection. However, it may be useful for providing relevant information on people’s immunoreaction to COVID-19 exposure.

"Usage of Silver-Stained Polyacrylamide Gels Electrophoresis (PAGE) for Detection of Rotavirus Infection by Direct Identification of Viral Nucleic Acid ."

Background: Rotavirus infects almost all children by the age of five. More than 180,000 annual deaths due to rotavirus, occurs in Bangladesh.Aims: This study aimed to determine the incidence of rotavirus infection in children by a modified polyacrylamide gel elctrophoretic (PAGE) in stool samples.Materials and Methods: In this descriptive type cross sectional study, a total of 400 stool samples were examined for the presence of rotavirus by a modified PAGE analysis of viral genome. Stool culture was done for common enteric pathogens. The study was carried out from November 2012 to July 2013 in the department of Microbiology, Mymensingh Medical College, Mymensingh.Results: PAGE results were found in 365 of 400 (91%) specimens. Maximum incidence of rotavirus infection was seen in age group of 6 months - 24 months (67.25%). All 151 rotavirus positive cases did not show infection with bacterial pathogens.Conclusion: The modified PAGE technique for the detection of viral RNA was found to be rapid, simple, reliable and less expensive technique.KYAMC Journal Vol. 6, No.-1, Jul 2015, Page 557-561

Usage of Silver-Stained Polyacrylamide Gels Electrophoresis (PAGE) for Detection of Rotavirus Infection by Direct Identification of Viral Nucleic Acid

Background: Rotavirus infects almost all children by the age of five. More than 180,000 annual deaths due to rotavirus, occurs in Bangladesh. Aims: This study aimed to determine the incidence of rotavirus infection in children by a modified polyacrylamide gel electrophoresis (PAGE) in stool samples. Materials and Methods: In this descriptive type cross sectional study, a total of 400 stool samples were examined for the presence of rotavirus by a modified PAGE analysis of viral genome. Stool culture was done for common enteric pathogens. The study was carried out from November 2012 to July 2013 in the department of Microbiology, Mymensingh Medical College, Mymensingh. Results: PAGE results were found in 365 of 400 (91%) specimens. Maximum incidence of rotavirus infection was seen in age group of 6 months - 24 months (67.25%). All 151 rotavirus positive cases did not show infection with bacterial pathogens.Conclusion: The modified PAGE technique for the detection of viral RNA was found to be rapid, simple, reliable and less expensive technique.Bangladesh Journal of Medical Science Vol.15(2) 2016 p.154-159

Recent Advances in Dengue Diagnosis

Dengue virus is a mosquito-borne flavivirus that causes a spectrum of illness ranging from asymptomatic infection to dengue fever to dengue haemorrhagic fever or dengue shock syndrome. Dengue virus has four distinct serotypes: DEN-1, DEN-2, DEN-3 and DEN-4. Dengue virus is endemic in many parts of the world, where it is responsible for significant morbidity and mortality. Laboratory confirmation of dengue infection relies on isolation of the virus in cell culture, identification of viral nucleic acid or antigens, detection of virus specific antibodies or a combination of these techniques. Timing and appropriate use of these diagnostic assays has to be understood.

Slow, persistent replication of lentiviruses: role of tissue macrophages and macrophage precursors in bone marrow.

Lentiviruses, as exemplified by visna virus of sheep, are nononcogenic retroviruses that cause slowly progressive diseases after prolonged periods of incubation. Earlier studies on visna have shown that the long incubation period of the disease is associated with constant production of minimal quantities of virus in tissues, whereas virus could be obtained by culturing monocytes and macrophages from explants of lymphatic tissues and inflamed organs. In this study the role of macrophages in lentivirus infection was explored using two sheep that were intrabronchially inoculated with virus. When sections of paraffin-embedded tissue, processed by a recently described technique which combines immunocytochemistry for the identification of macrophages and in situ hybridization for identification of viral nucleic acid, were examined, we found that virus replication is associated almost exclusively with infection in selected populations of macrophages in the interalveolar region of the alveoli, in inflammatory exudate cells in the lung, in lymph nodes, and in the spleen. Although large numbers of alveolar macrophages had viral RNA, few of these cells produced virus. While this minimally productive type of viral replication provides an explanation for the slow pace of the infection, restricted replication in terminally differentiated, short-lived macrophages does not explain persistent virus replication in the animal. With the discovery of clusters of infected macrophage precursors in the bone marrow, a mechanism for persistence was found. The macrophage precursor cells provide an important missing link in the virus-target-cell circuit and may be the reservoir of latently infected cells which perpetuate lentivirus infections in both animals and humans.

Staining of Bacteriophage Nucleic Acids with Acridine Orange

THE use of the fluorescent stain acridine orange for the quick identification of viral nucleic acids has been described by Mayor and Hill1. In this valuable technique colour differences permit differentiation between double-stranded deoxyribonucleic acid (DNA), on one hand (fluoresces green), and ribonucleic acid (RNA) and single-stranded DNA on the other (fluoresces red). The latter nucleic acids can be separated by their respective sensitivities to RNase and DNase. While the procedure of Mayor and Hill is simple compared with the techniques of biochemical analysis, it involves numerous treatments and the use of fluorescence microscopy. Furthermore, acridine orange staining is particularly sensitive to pH levels, and, because of this, some difficulty may be experienced in obtaining the correct colours. In addition, the procedure does not differentiate between single-stranded DNA and RNA, using the stain alone. This communication describes some improvements and modifications with these points in mind. Direct viewing under ultra-violet light is substituted for the fluorescence microscope, the pre-staining treatment is simplified, and possible unreliability due to pH sensitivity is eliminated.