Virus RNA Quantification Research Articles

Age-related differences in immune dynamics during SARS-CoV-2 infection in rhesus macaques.

Advanced age is a key predictor of severe COVID-19. To gain insight into this relationship, we used the rhesus macaque model of SARS-CoV-2 infection. Eight older and eight younger macaques were inoculated with SARS-CoV-2. Animals were evaluated using viral RNA quantification, clinical observations, thoracic radiographs, single-cell transcriptomics, multiparameter flow cytometry, multiplex immunohistochemistry, cytokine detection, and lipidomics analysis at predefined time points in various tissues. Differences in clinical signs, pulmonary infiltrates, and virus replication were limited. Transcriptional signatures of inflammation-associated genes in bronchoalveolar lavage fluid at 3 dpi revealed efficient mounting of innate immune defenses in both cohorts. However, age-specific divergence of immune responses emerged during the post-acute phase. Older animals exhibited sustained local inflammatory innate responses, whereas local effector T-cell responses were induced earlier in the younger animals. Circulating lipid mediator and cytokine levels highlighted increased repair-associated signals in the younger animals, and persistent pro-inflammatory responses in the older animals. In summary, despite similar disease outcomes, multi-omics profiling suggests that age may delay or impair antiviral cellular immune responses and delay efficient return to immune homeostasis.

Renal pathological changes in sheep infected with BTV-1 during early pregnancy

Bluetongue is a vector borne, non-contagious, viral disease of ruminants affecting many species of ruminants including cattle, buffalo, sheep, goats, deer, antelope and camel etc. BTV replication and lesions were reported in tissues and organs including skin, lymph nodes, spleen, bone marrow, liver, heart etc. BTV induced lesions have also been observed in the kidney of sheep that include petechiae, subcapsular haemorrhages, and renal tubular necrosis. However, the effect of BTV-1 in the kidney of pregnant sheep remains elusive as pathogenesis of the BTV varied with the serotype and species of the animals. The goal of the present study was to study BTV-1 induced pathological changes in the kidney and viral RNA quantification in pregnant sheep those experimentally infected during early pregnancy. For this, kidney from the sacrificed animals were collected in 10% neutral buffered formalin and RNA later. The presence of the Viral RNA and viral antigen in the ovine kidney and other organs samples was investigated at different day post infection. Histopathological examination of kidneys revealed hypercellularity of mesangial cells, degeneration of renal tubules, casts and multifocal infiltration of lymphocytes and changes were more pronounced at 15th dpi. None of the kidney tissue from infected sheep revealed the BTV antigen using immunohistochemistry. Thus, we concluded that experimental infection of sheep with BTV during early pregnancy induces pathological changes in the kidneys and also leads to moderate viral load in different organs. Further, BTV serotype 1 is unlikely to contribute directly in the renal pathology of pregnant sheep.

HCV RNA quantification in capillary dried blood spots with the Xpert® HCV Viral Load test for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection.

For patients with difficult venous access after long-term intravenous drug use, rapid point-of-care hepatitis C virus (HCV) RNA quantification in capillary whole blood with the Xpert® HCV Viral Load Fingerstick (VL FS) test (60 minutes) is a convenient and reliable method for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection. However, an expensive GeneXpert® system must be available on site. In decentralised settings with a low case-load, dried blood spot (DBS) testing might be an alternative. Between December 2019 and January 2021, patients with an indication for HCV RNA quantification and informed consent provided 100 µl capillary whole blood each for on-site Xpert® HCV VL FS testing (reference) and DBS testing in the laboratory. For the latter, 100 µl blood, collected with an EDTA Minivette®, were transferred to a Whatman® 903 filter card. After drying for at least 1 hour, the DBS sample was packed into a sealable plastic bag with desiccant and sent to the central laboratory of our hospital, where it was stored at -20°C. For HCV RNA extraction, the whole DBS was cut out with an 18-mm puncher and transferred into 1.3 ml guanidinium thiocyanate-containing buffer (provided by Cepheid®). After mixing and incubating at room temperature for 2-3 hours, 1 ml supernatant was analysed with the Xpert® HCV VL test (105 minutes) (filter paper absorbs 0.3 ml). Of 109 paired samples from 67 patients, 38 (34.9%) were positive with the Xpert® HCV VL FS test. Sensitivity and specificity of DBS testing were 89.5% (34/38; 95% confidence interval [CI] 75.9-95.8%) and 97.2% (69/71; 95% CI 90.3-99.2%), respectively. The six (5.5%) discordant results (four false negative, two false positive) all were observed in samples with HCV RNA detectable below the limit of quantification after 2-8 weeks of pan-genotypic direct-acting antiviral treatment or 5 weeks after acute hepatitis C in a patient clearing HCV spontaneously. Quantifiable results (n = 30; 16 genotype 1, 7 genotype 3, 4 genotype 4, 1 genotype 1a and 3a, 2 unknown; HCV RNA range: 2.74-6.66 log IU/ml) correlated well (R2 = 0.981). On average, uncorrected DBS test results were 1.30 ± 0.14 log IU/ml lower than Xpert® HCV VL FS test results (~42 μl instead of the expected 1000 μl plasma used). Storage of DBS samples at room temperature for 7 days before freezing reduced HCV RNA by 0.29 ± 0.12 log IU/ml. HCV RNA can reliably be quantified with the Xpert® HCV VL test in capillary dried blood spot samples. Thus, access to capillary HCV RNA quantification for diagnosing chronic HCV infection, monitoring treatment and detecting reinfection can be extended to decentralised settings with a low case load.

Digital CRISPR/Cas12b-based platform enabled absolute quantification of viral RNA

ABSTRACT: Early and accurate diagnosis of viruses is critical for control of the pandemic. CRISPR/Cas-based detection of nucleic acid is an emerging technology for molecular diagnostics, and has been applied for virus detection. Though these methods have excellent sensitivity and specificity, most of them were not able to measure the quantity of virus. We here developed a droplet digital reverse transcription loop-mediated isothermal amplification (RT-LAMP) enhanced Cas12b-based RNA detection platform (RECD), for quantitative detection of viral RNA. CRISPR/Cas12b, which is more thermally stable than other family members in CRISPR systems, is combined with digital RT-LAMP. Due to the innate characteristic of digital format detection and CRISPR/Cas system, droplet digital RECD (ddRECD) assay enables absolute quantification of viral RNA, with single-molecule sensitivity. We expect the ddRECD assay will be a powerful tool for molecular diagnostics.

Digital Quantification of Human Viral RNA and DNA Using a Self-Digitization Chip.

Digital nucleic acid quantitation methods show excellent sensitivity and specificity for pathogen detection. Droplet digital PCR (ddPCR) is the most advanced digital nucleic acid quantitation method and has been commercialized, but is not suitable for many point-of-care applications due to its complex instrumentation. Here we describe a simple microfluidics-based self-digitization (SD) chip for quantifying nucleic acids at the point of care with minimal instrumentation. We demonstrate the clinical diagnostic capability of this platform by applying it to quantifying human viral DNA and RNA. SD chips with a range of well numbers and volumes are tested, and isothermal methods are used to amplify the DNA and RNA to a detectable level. Sample concentration is determined based on the measured volume in the wells and the number of wells with fluorescence greater than a threshold based on a Poisson distribution. Concentration measurements over the low concentration range of 0-100molecules/μL showed a strong correlation (R2=0.99) with measurements using a real-time PCR assay, demonstrating the sensitivity and specificity of the SD chip platform.

Innovative Toolbox for the Quantification of Drosophila C Virus, Drosophila A Virus, and Nora Virus

Quantification of viral replication underlies investigations into host-virus interactions. In Drosophila melanogaster, persistent infections with Drosophila C virus, Drosophila A virus, and Nora virus are commonly observed in nature and in laboratory fly stocks. However, traditional endpoint dilution assays to quantify infectious titers are not compatible with persistently infecting isolates of these viruses that do not cause cytopathic effects in cell culture. Here we present a novel assay based on immunological detection of Drosophila C virus infection that allows quantification of infectious titers for a wider range of Drosophila C virus isolates. We also describe strand specific RT-qPCR assays for quantification of viral negative strand RNA produced during Drosophila C virus, Drosophila A virus, and Nora virus infection. Finally, we demonstrate the utility of these assays for quantification of viral replication during oral infections and persistent infections with each virus.

SARS-CoV-2 Blood RNA Load Predicts Outcome in Critically Ill COVID-19 Patients

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with coronavirus disease 2019 (COVID-19).MethodsWe evaluated the predictive value of viral RNA loads and courses in the blood compared with the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by reverse transcription quantitative polymerase chain reaction were performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire intensive care unit (ICU) stay (4145 samples analyzed). Patients were grouped according to their 90-day outcome as survivors (n=100) or nonsurvivors (n=70).ResultsIn nonsurvivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (P=.0009). Failure of blood RNA clearance was observed in 33/50 (66%) of the nonsurvivors compared with 12/64 (19%) survivors (P<.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (odds ratio [OR; log10], 0.23; 95% CI, 0.12–0.42; P<.0001), with a significantly higher effect for survival compared with respiratory tract SARS-CoV-2 RNA loads (OR [log10], 0.75; 95% CI, 0.66–0.85; P<.0001). Blood RNA loads exceeding 2.51×103 SARS-CoV-2 RNA copies/mL were found to indicate a 50% probability of death. Consistently, 29/33 (88%) nonsurvivors with failure of virus clearance exceeded this cutoff value constantly.ConclusionsBlood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.

Human immunodeficiency virus type 1 (HIV-1) subtype diversity in Busia, Western Kenya

HIV infection is currently the single biggest epidemic globally. HIV the etiologic agent for AIDS is divided into two types: HIV-1 and HIV-2. HIV-2 is rare and is mainly found in some parts of West Africa. HIV-1 accounts for most cases of AIDS reported globally. HIV-1 strains can be classified into four groups: The major group M, group O, group N and the most recent group P. All of which may represent separate introductions of SIVs into humans. This cross sectional study determined the HIV-1 subtype diversity in Busia, Western Kenya. Briefly, participants were consented into the study based on pre-determined inclusion criteria. Viral RNA quantification was performed to select participants with virologic failure for drug resistance testing. HIV drug resistance testing (DRT) was performed and sequences obtained were used to determine circulating HIV-1 subtypes using the REGA HIV-1 Subtyping Tool Version 3.0. Phylogenetic analysis was performed using MEGA software V7.0 to confirm the circulating HIV subtypes. Out of 915 participants screened, 146 participants had virologic failure although 140 were successfully sequenced. Subtype A1 was the most prevalent subtype present in 52.9% of the participants followed by subtype D (20.7%), CRF A1_D (7.1%) subtype C and subtype B (4.3%) and subtype A2 (3.6%). Sequences within the same subtype and CRF clustered close together on the phylogenetic tree. An increase in CRFs in the population compared to previous studies. Circulating HIV subtypes should be continually monitored in Busia to determine trends in transmission and map the circulating recombinant forms for epidemiological purposes. Key words: HIV-1, Busia county, subtype diversity, reverse transcriptase.

Quantification of Viral RNA in Multiple Pieces of Explant Liver Tissue Shows Distinct Focal Differences in Hepatitis B Infection.

Hepatitis B virus (HBV) DNA and RNA were quantified by digital PCR assays in 20–30 tissue pieces from each of 4 liver explants with cirrhosis caused by HBV. The within-patient variability of HBV RNA levels between pieces was up to a 1000-fold. Core RNA and S RNA levels were similar and correlated strongly when replication was high, supporting that transcription was from covalently closed circular DNA (cccDNA). By contrast, enhanced expression of S RNA relative to cccDNA and core RNA in patients with medium-high or low replication supports that HBV surface antigen (HBsAg) can be expressed mainly from integrated HBV DNA in such patients.

A standardized definition of placental infection by SARS-CoV-2, a consensus statement from the National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development SARS-CoV-2 Placental Infection Workshop

Pregnant individuals infected with SARS-CoV-2 have higher rates of intensive care unit admission, oxygen requirement, need for mechanical ventilation, and death than nonpregnant individuals. Increased COVID-19 disease severity may be associated with an increased risk of viremia and placental infection. Maternal SARS-CoV-2 infection is also associated with pregnancy complications such as preeclampsia and preterm birth, which can be either placentally mediated or reflected in the placenta. Maternal viremia followed by placental infection may lead to maternal-fetal transmission (vertical), which affects 1% to 3% of exposed newborns. However, there is no agreed-upon or standard definition of placental infection. The National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health and Human Development convened a group of experts to propose a working definition of placental infection to inform ongoing studies of SARS-CoV-2 during pregnancy. Experts recommended that placental infection be defined using techniques that allow virus detection and localization in placental tissue by one or more of the following methods: in situ hybridization with antisense probe (detects replication) or a sense probe (detects viral messenger RNA) or immunohistochemistry to detect viral nucleocapsid or spike proteins. If the abovementioned methods are not possible, reverse transcription polymerase chain reaction detection or quantification of viral RNA in placental homogenates, or electron microscopy are alternative approaches. A graded classification for the likelihood of placental infection as definitive, probable, possible, and unlikely was proposed. Manuscripts reporting placental infection should describe the sampling method (location and number of samples collected), method of preservation of tissue, and detection technique. Recommendations were made for the handling of the placenta, examination, and sampling and the use of validated reagents and sample protocols (included as appendices).

Systematic analysis of SARS-CoV-2 infection of an ACE2-negative human airway cell.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) variants govern transmissibility, responsiveness to vaccination, and disease severity. In a screen for new models of SARS-CoV-2 infection, we identify human H522 lung adenocarcinoma cells as naturally permissive to SARS-CoV-2 infection despite complete absence of angiotensin-converting enzyme 2 (ACE2) expression. Remarkably, H522 infection requires the E484D S variant; viruses expressing wild-type S are not infectious. Anti-S monoclonal antibodies differentially neutralize SARS-CoV-2 E484D S in H522 cells as compared to ACE2-expressing cells. Sera from vaccinated individuals block this alternative entry mechanism, whereas convalescent sera are less effective. Although the H522 receptor remains unknown, depletion of surface heparan sulfates block H522 infection. Temporally resolved transcriptomic and proteomic profiling reveal alterations in cell cycle and the antiviral host cell response, including MDA5-dependent activation of type I interferon signaling. These findings establish an alternative SARS-CoV-2 host cell receptor for the E484D SARS-CoV-2 variant, which may impact tropism of SARS-CoV-2 and consequently human disease pathogenesis.

SARS-CoV-2 complete genome sequencing from the Italian Campania region using a highly automated next generation sequencing system

BackgroundSince the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus.MethodsRNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March–April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software.ResultsThe complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes.ConclusionsThe Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.

Infection of zebrafish larvae with human norovirus and evaluation of the in vivo efficacy of small-molecule inhibitors.

We have recently established that human norovirus (HuNoV) replicates efficiently in zebrafish larvae after inoculation of a clinical sample into the yolk, providing a simple and robust in vivo system in which to study HuNoV. In this Protocol Extension, we present a detailed description of virus inoculation by microinjection, subsequent daily monitoring and harvesting of larvae, followed by viral RNA quantification. This protocol can be used to study viral replication of genogroup (G)I and GII HuNoVs in vivo within 3-4 d. Additionally, we describe how to evaluate the in vivo antiviral effect and toxicity of small molecules using HuNoV-infected zebrafish larvae, in multi-well plates and without the need for specific formulations. This constitutes a great advantage for drug discovery efforts, as no specific antivirals or vaccines currently exist to treat or prevent norovirus gastroenteritis.

Real-time PCR assay development for the control of vaccine against hemorrhagic fever with renal syndrome

Hemorrhagic fever with renal syndrome (HFRS) holds a leading place among natural focal human diseases in Russian Federation. There is no etiotropic therapy for the disease now. The vaccine prophylaxis is the most effective method to control this infection. The main criteria for inactivated vaccines evaluation are its immunogenicity and specific activity.The study purposes were to develop a sensitive and specific real-time PCR method for viral RNA quantification in the inactivated vaccine and to study the correlation between the viral RNA amount and vaccine immunogenicity. L-segment fragments of the Puumala, Hantaan, and Sochi vaccine strains were selected as diagnostic targets for oligonucleotides and fluorescent probes. The immunogenicity of experimental vaccines was determined by the induction of neutralizing antibodies in BALB/c mice. A highly specific, sensitive and reproducible real-time PCR method has been developed. The analytical sensitivity was 1.24 ± 1.5 x 102 copies/ml for Puumala virus; 1.16 ± 1.4 * 102 copies/ml for Hantaan; 1.32 ± 1.8 * 102 copies/ ml for Sochi, with a virus content of 1.5 ± 0.5 lg FFU/ml; 1.8 ± 0.5 lg FFU/ml and 2.2 ± 0.5 lg FFU/ml, respectively. The viral RNA amount in experimental vaccine preparations inactivated with β-propiolactone was proportional to the neutralizing antibodies titer observed in mice following the immunization. It was found that different virus inactivators differently affects the detected viral RNA amount, but not the vaccine immunogenicity, which indicates the same degree of the immunogenic proteins damage. The direct relationship between the viral RNA copy number and vaccine immunogenicity makes it possible to use this criterion for vaccine dosage preparation. The developed method for viral RNA quantification is a promising tool for the specific activity control of the HFRS vaccine.

Performances of NeuMoDx™, a random-access system for hepatitis B virus DNA and hepatitis C virus RNA quantification

ObjectiveMonitoring of viral loads (VL) for hepatitis B and C viruses (HBV; HCV) is essential to evaluate disease progression and treatment response. Automated, random-access rapid systems are becoming standard to provide clinicians with reliable VL. The aim of this study was to evaluate the analytical performances of the recently launched NeuMoDx™ for HBV-DNA and HCV-RNA quantification. MethodsClinical samples routinely quantified on the Beckman–Veris system were either retrospectively (frozen samples; HBV n = 178, HCV n = 249), or in parallel (fresh primary tubes; HBV n = 103, HCV n = 117) tested using NeuMoDx™. Linearity range was assessed on serial dilutions of high-titre plasmas containing different genotypes for HBV (A–E, n = 9) and HCV (1a,1b,2–5, n = 12). ResultsOverall test failure, mostly internal control amplification failure, was 2.3% and was not influenced by matrix types (fresh or frozen). For HBV VL, κ agreement was 74%, with 27 (12.6%) discrepancies. Correlation between HBV assays on 72 quantified samples by both methods was excellent (r = 0.963) with a mean bias (NeuMoDx™–Veris) of 0.21 log IU/mL. For HCV VL, κ agreement reached 94%, with 9 (2.8%) discrepancies. The r correlation factor between assays on 104 samples was 0.960 with a mean bias of –0.14 log IU/mL (NeuMoDx™–Veris). Serial dilutions confirmed the claimed linear ranges for all analysed HBV and HCV genotypes. The mean turnaround time was 72 minutes (range 55–101 minutes) for HBV and 96 minutes (range 78–133 minutes) for HCV. ConclusionResults obtained on the NeuMoDx™ confirmed the overall good functionality of the system with a short turn-around-time, full traceability and easy handling. These results on HBV and HCV VL look promising and should be challenged with further comparisons.

Human Respiratory Syncytial Virus Infection in a Human T Cell Line Is Hampered at Multiple Steps.

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory disease in children. The main targets of HRSV infection are epithelial cells of the respiratory tract, and the great majority of the studies regarding HRSV infection are done in respiratory cells. Recently, the interest on respiratory virus infection of lymphoid cells has been growing, but details of the interaction of HRSV with lymphoid cells remain unknown. Therefore, this study was done to assess the relationship of HRSV with A3.01 cells, a human CD4+ T cell line. Using flow cytometry and fluorescent focus assay, we found that A3.01 cells are susceptible but virtually not permissive to HRSV infection. Dequenching experiments revealed that the fusion process of HRSV in A3.01 cells was nearly abolished in comparison to HEp-2 cells, an epithelial cell lineage. Quantification of viral RNA by RT-qPCR showed that the replication of HRSV in A3.01 cells was considerably reduced. Western blot and quantitative flow cytometry analyses demonstrated that the production of HRSV proteins in A3.01 was significantly lower than in HEp-2 cells. Additionally, using fluorescence in situ hybridization, we found that the inclusion body-associated granules (IBAGs) were almost absent in HRSV inclusion bodies in A3.01 cells. We also assessed the intracellular trafficking of HRSV proteins and found that HRSV proteins colocalized partially with the secretory pathway in A3.01 cells, but these HRSV proteins and viral filaments were present only scarcely at the plasma membrane. HRSV infection of A3.01 CD4+ T cells is virtually unproductive as compared to HEp-2 cells, as a result of defects at several steps of the viral cycle: Fusion, genome replication, formation of inclusion bodies, recruitment of cellular proteins, virus assembly, and budding.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BackgroundCovid-19 clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during Covid-19 and if monocytes and macrophages could be infected by SARS-CoV-2.MethodsMonocytes and monocyte-derived macrophages from Covid-19 patients and controls were infected with SARS-CoV-2, and extensively investigated with immunofluorescence, viral RNA extraction and quantification, total RNA extraction followed by reverse transcription and q-PCR using specific primers, supernatant cytokines (IL-10, TNF-α, IL-1β, IFN-β, TGF-β1 and IL-6), flow cytometry. The effect of M1- versus M2-type or no polarization prior to infection was assessed.ResultsSARS-CoV-2 efficiently infected monocytes and MDMs but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In Covid-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity.ConclusionSARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of Covid-19 progression.

Determination of FMDV 146S particle concentration by spectrometric method during viral RNA quantification

During FMD vaccine production, special attention is paid to the concentration of 146S particles bearing the critical biological features of FMDV and being the main components that have an effect on vaccine immunogenicity. For this reason, each batch of vaccine raw material is tested for 146S component concentration. The paper presents the results of the use of a spectrometric method for whole particle concentration determination during quantification of FMDV RNA extracted after immune capture. It is an inexpensive, easy-to-perform method allowing for determination of FMDV 146S particle concentration in the non-inactivated culture suspension. 146S particle concentration was found to depend on the number of RNA molecules extracted from virions after their strain-specific immune capture and quantitatively detected by the spectrometric method. The presented method allows for determination of 146S component concentration in the non-inactivated vaccine raw material using the proposed linear model. The spectrometric method showed 94.5–99.5% correlation with real time reverse transcription polymerase chain reaction and complement fixation test based on the results of tests of 360 non-inactivated suspensions of FMDV of all types. Tests of the positive control demonstrated 99.0–99.6% compatibility of actual and expected results. FMDV genome and 146S particles were not detected in the negative control, and that was in line with expectations.

Chlorhexidine: An effective anticovid mouth rinse.

Context:Dentists across the globe are witnessing a completely unforeseen and uncertain professional situation during these times of COVID-19 pandemic. There is conflicting evidence regarding the effectiveness of routinely used mouthwashes and especially Chlorhexidine, to reduce the viral load in oral cavity and the aerosols during oral procedures.Aims:Comparative evaluation of the effectiveness of the current 'gold standard' chlorhexidine and povidone iodine as a control agent, through an in-vitro analysis.Settings and Design:In-vitro laboratory analysis.Methods and Material:All the experiments for analysis of antiviral efficacy of chlorhexidine digluconate (2%)and povidone iodine(1%), against SARS-CoV-2 virus were performed in the BSL3 facility at the Council of Scientific and Industrial Research-Institute of Microbial Technology, using the VeroE6 cell lines. The analysis of the virus inactivation was based on quantification of viral RNA (Cycle threshold (Ct) profile) present in the culture supernatant using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR).Statistical Analysis Used:Descriptive analysis (Statistical package for social sciences (SPSS Inc., Chicago, IL, version 15.0 for Windows).Results:Chlorhexidine digluconate in 0.2% concentration inactivated more than 99.9% of SARS CoV 2 virus, in minimal contact time of 30 seconds, which was considered better efficacy than povidone-iodine utilized for 30 and 60 seconds. Subtle differences were observed in the activity of both the compounds in terms of percent inactivation of virus, though a greater relative change in Ct values was observed for chlorhexidine.Conclusions:Within the limitations of the present study, it can be concluded that Chlorhexidine digluconate in 0.2% concentration inactivated SARS CoV 2 in minimal contact time i.e 30 secs, however both compounds tested i.e Chlorhexidine and povidone-iodine were found to have antiviral activity against SARS CoV2 virus.

Infection by Zika Vírus in human cells alters the expression profile of miRNA-15 and activation of apoptotic caspases

Objective: Evaluate the miRNA-15 expression profile involved in cellular apoptotic regulation factors. Methodology: We used the H818308 Asian strain of ZIKV without neurological damage. The inoculations occurred in human embryonic kidney cells (HEK-293). After inoculation, samples were extracted for RT-qPCR quantification of viral RNA and miR-15. The level of activation of caspases 1, 3/7 and 8 of cells was performed using chemofluorescence. Results: The ZIKV infection alters the expression of genes and their regulators, affecting several cellular physiological processes such as apoptosis. Conclusion: Therefore, it is important to emphasize that renal progenitor cells (HEK-293) are susceptible to VZIK infection. The genetic deregulation resulting from infection directly affects important cellular processes such as apoptosis from the disordered miRNA-15 expression during the infection period.