Virus Samples Research Articles

Isolamento e avaliação do comportamento do vírus ORF em células de córnea fetal caprina e identificação pela Reação em Cadeia da Polimerase

The control of the infection in endemic regions is performed with vaccines, however there are limitations in the vaccine production due to the difficulties in replicating the virus in cell cultures. This work was conducted so as to isolate and evaluate the behavior of ECV samples in fetal goat cell line cornea (CorFC) naturally immortalized. Crust samples from 22 sheep and seven goats presenting the clinical symptoms of EC from the states of Pernambuco, Bahia, Sergipe and Paraíba, were inoculated in CorFC monolayers and identified by polymerase chain reaction (PCR) using primers for amplification of a fragment of235 bp of gene B2L envelope ECV. Eleven samples were submitted to seven consecutive passes, at weekly intervals. Cytopathic effect (CPE) was observed in all passages, after24 hours post-infection, characterized by cell rounding, syncytial fusion with training, inclusion and cytoplasmic vacuolization. Thus, CorFC cell cultures proved highly permissible to ECV replication with small variation among viral samples. The PCR technique can be an efficient method used for confirmation of ECV infection in clinical samples.

Evaluation of potassium peroxymonosulfate (MPS) efficacy against SARS-CoV-2 virus using RT-qPCR-based method

The SARS-CoV-2 virus spreads mostly through the respiratory droplet and contaminated fomites has been proposed as possible viral transmission. Several disinfectants were previously recommended for SARS-CoV-2 decontamination including alcohol, hydrogen peroxide, and sodium hypochlorite with some limitations. Potassium peroxymonosulfate (MPS) is a broadspectrum disinfectants and acts on bacterial and viral protein capsids by oxidation. It is widely used as multipurpose virucidal disinfection in livestock industry. However, there is no direct evidence shows MPS is effective against the SARS-CoV-2 virus. SARS-CoV-2 virus positive samples from Thai patients in vital transport medium were treated with MPS at different dilutions (1:100, 1:200, 1:400) with various contact times (1, 5, 10 minutes).Sodium hypochlorite was included as a reference disinfectant against SARS-CoV-2 virus. Subsequently, viral genomic RNA samples were then extracted and performed SARS-CoV-2 Real-time, PCR by targeting viral ORF1ab and N genes to detected SARS-CoV-2 genomic RNA integrity. Using triplicate experiments, the results indicated that all 3 MPS dilutions (1:100/1:200/1:400) statistically reduced the SARS-CoV-2 genome integrity resulting in reduction of SARS-CoV-2 detection by 93.7%, 88.7%, and 83.2%, respectively based on the ORF1ab gene compared to negative control. Sodium hypochlorite (1%) induced > 99.9% loss in the SARS-CoV-2 detection. Similar results were observed by the N gene amplification. The internal control of all RT-qPCR reactions was not significantly different. Our results provide the first evidence that MPS has a disinfected efficacy against the SARS-CoV-2 virus and could be used as an environmental decontaminant in the household, hospital facilities and food industry with an acceptable safety profile.

Evaluation of stability and inactivation methods of SARS-CoV-2 in context of laboratory settings

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Laboratory work with SARS-CoV-2 in a laboratory setting was rated to biosafety level 3 (BSL-3) biocontainment level. However, certain research applications in particular in molecular biology require incomplete denaturation of the proteins, which might cause safety issues handling contaminated samples. In this study, we evaluated lysis buffers that are commonly used in molecular biological laboratories for their ability to inactivate SARS-CoV-2. In addition, viral stability in cell culture media at 4 °C and on display glass and plastic surfaces used in laboratory environment was analyzed. Furthermore, we evaluated chemical and non-chemical inactivation methods including heat inactivation, UV-C light, addition of ethanol, acetone-methanol, and PFA, which might be used as a subsequent inactivation step in the case of insufficient inactivation. We infected susceptible Caco-2 and Vero cells with pre-treated SARS-CoV-2 and determined the tissue culture infection dose 50 (TCID50) using crystal violet staining and microscopy. In addition, lysates of infected cells and virus containing supernatant were subjected to RT-qPCR analysis. We have found that guanidine thiocyanate and most of the tested detergent containing lysis buffers were effective in inactivation of SARS-CoV-2, however, the M-PER lysis buffer containing a proprietary detergent failed to inactivate the virus. In conclusion, careful evaluation of the used inactivation methods is required especially for non-denaturing buffers. Additional inactivation steps might be necessary before removal of lysed viral samples from BSL-3.

COVID-19: 2020 VISION

Several months into 2020, the COVID-19 outbreak was described as a wake-up call regarding the risk of a novel coronavirus pandemic. In fact, SARS-CoV-2 was that pandemic virus, and the pandemic was already underway. Australia responded comparatively quickly and effectively with detection of the initial community incursions by SARS-CoV-2, and standing-up a coordinated national laboratory response. The scale of testing undertaken since then has been unprecedented. It has entailed some unique challenges, such as supply chain insecurity, workplace restrictions, and support for quarantine hotel isolation through high-frequency testing of staff and isolated travellers. Innovative approaches have been required. The application and scale-up of some emerging technologies and technical approaches have been fast-tracked, including viral genomics, sample pooling, and saliva-based surveillance testing. The development and roll-out of vaccines has been an enormous technical achievement, and offers hope for mitigation. However, the potential threat posed by unexpected amounts of virus mutation, producing so-called ‘variants of concern’, remains as yet imperfectly understood. I believe that we can be guardedly optimistic about the future in Australasia, however there is as yet much road to be travelled.

Fitness Estimation for Viral Variants in the Context of Cellular Coinfection.

Animal models are frequently used to characterize the within-host dynamics of emerging zoonotic viruses. More recent studies have also deep-sequenced longitudinal viral samples originating from experimental challenges to gain a better understanding of how these viruses may evolve in vivo and between transmission events. These studies have often identified nucleotide variants that can replicate more efficiently within hosts and also transmit more effectively between hosts. Quantifying the degree to which a mutation impacts viral fitness within a host can improve identification of variants that are of particular epidemiological concern and our ability to anticipate viral adaptation at the population level. While methods have been developed to quantify the fitness effects of mutations using observed changes in allele frequencies over the course of a host’s infection, none of the existing methods account for the possibility of cellular coinfection. Here, we develop mathematical models to project variant allele frequency changes in the context of cellular coinfection and, further, integrate these models with statistical inference approaches to demonstrate how variant fitness can be estimated alongside cellular multiplicity of infection. We apply our approaches to empirical longitudinally sampled H5N1 sequence data from ferrets. Our results indicate that previous studies may have significantly underestimated the within-host fitness advantage of viral variants. These findings underscore the importance of considering the process of cellular coinfection when studying within-host viral evolutionary dynamics.

Emtricitabine triphosphate in dried blood spots predicts future viremia in persons with HIV and identifies mismatch with self-reported adherence.

Emtricitabine triphosphate (FTC-TP) in dried blood spots (DBS), a measure of short-term antiretroviral therapy (ART) adherence, is associated with viral suppression in persons with HIV (PWH). However, its ability to predict future viremia remains unknown. Prospective, observational cohort (up to three visits in 48 weeks). PWH receiving TDF/FTC-based ART had DBS and HIV viral load obtained at routine clinical visits. FTC-TP in DBS was dichotomized into quantifiable vs. below the limit of quantification (BLQ). The adjusted odds ratio (aOR) of future viremia (≥20 copies/ml at next study visit) was estimated according to FTC-TP at the current visit. To assess for possible interactions, additional models adjusted for tenofovir diphosphate (TFV-DP) in DBS and 3-day self-reported adherence. Data from 433 PWH (677 paired DBS/HIV viral load samples) were analyzed. The aOR [95% confidence interval (CI)] for future viremia for BLQ vs. quantifiable FTC-TP was 3.4 (1.8--6.5; P = 0.0002). This diminished after adjusting for TFV-DP [aOR 1.9 (0.9--4.1); P = 0.090]. Among PWH reporting 100% 3-day adherence, the odds of future viremia were 6.0 times higher [(1.8--20.3); P = 0.001] when FTC-TP was BLQ vs. quantifiable. Among participants (n = 75) reporting less than 100% adherence, BLQ FTC-TP in DBS was not predictive of future viremia [aOR 1.3 (0.4--4.6); P = 0.96]. Nonquantifiable FTC-TP in DBS predicts future viremia and is particularly informative in PWH reporting perfect adherence. As point-of-care adherence measures become available, mismatches between objective and subjective measures, such as FTC-TP in DBS and self-report, could help clinicians identify individuals at an increased risk of future viremia.

Short Communication: Analysis of purity and concentration of DNA extracted from intron patho gene-spin extraction on crab processed food product samples

Abstract. Sophian A, Purwaningsih R, Muindar, Igirisa EPJ, Amirullah ML. 2021. Short Communication: Analysis of purity and concentration of DNA extracted from intron patho gene-spin extraction on crab processed food product samples. Asian J Trop Biotechnol 18: 28-31. The purpose of this study was to optimize the Intron patho gene-spin DNA/RNA extraction kit on crab processed food products so that it can be used for testing samples other than viral and bacterial samples. The method used in purity and concentration analysis to analyze total DNA (yield) is the absorbance method using a nanophotometer and the method used to isolate the sample DNA is the centrifuge Column or spin column method. The research data showed that the extracted sample concentration was in the range of 15.15-15.75, with an average of 15.46. while the purity values measured at the A260/A280 wavelengths were obtained with a purity range between 2,124-2,346. The total DNA analysis (yield) results obtained an average of 755.04 with the lowest yield value of 575.50 and the highest yield value of 787.50. As a suggestion for further research, it is hoped that modification can be made to perfect the isolated DNA so that it has better purity and concentration values.

Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Ultrasensitive Detection of SARS-CoV-2 in Saliva and Viral Transport Medium Clinical Samples.

The COVID-19 pandemic has underscored the shortcomings in the deployment of state-of-the-art diagnostics platforms. Although several polymerase chain reaction (PCR)-based techniques have been rapidly developed to meet the growing testing needs, such techniques often need samples collected through a swab, the use of RNA extraction kits, and expensive thermocyclers in order to successfully perform the test. Isothermal amplification-based approaches have also been recently demonstrated for rapid severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection by minimizing sample preparation while also reducing the instrumentation and reaction complexity. In addition, there are limited reports of saliva as the sample source, and some of these indicate inferior sensitivity when comparing reverse transcription loop-mediated isothermal amplification (RT-LAMP) with PCR-based techniques. In this paper, we demonstrate an improved sensitivity assay from saliva using a two-step RT-LAMP assay, where a short 10 min RT step is performed with only B3 and backward inner primers before the final reaction. We show that while the one-step RT-LAMP demonstrates satisfactory results, the optimized two-step approach allows detection of only few molecules per reaction and performs significantly better than the one-step RT-LAMP and conventional two-step RT-LAMP approaches with all primers included in the RT step. We show control measurements with RT-PCR, and importantly, we demonstrate RNA extraction-free RT-LAMP-based assays for detection of SARS-CoV-2 from viral transport media and saliva clinical samples.

Impact of RNA degradation on influenza diagnosis in the surveillance system

BackgroundThe continuous evolution of influenza viruses is monitored by the World Health Organization Global Influenza Surveillance and Response System. Sample quality is essential for surveillance quality. MethodsTo evaluate the RNA degradation of clinical samples, influenza-like illness samples were collected from four sentinel hospitals, and seasonal influenza was tested by real-time reverse transcription polymerase chain reaction and quantified by digital reverse transcription polymerase chain reaction at different time points. ResultsRNA degradation was observed in the majority of samples eight days after sample collection. A significant and faster rate of RNA content reduction was observed in low viral load samples (<10 copies/µl) than in high viral load samples (>10 copies/μl), stored at 2 to 8°C for up to eight days. RNase P (RNP) RNA, which is a key indicator to evaluate sample collection quality, was detected. Sample collection quality was uneven in different hospitals. ConclusionLow viral load samples increase the risk of false negatives due to RNA degradation to undetectable levels.

Pool testing on random and natural clusters of individuals: Optimisation of SARS-CoV-2 surveillance in the presence of low viral load samples.

Facing the SARS-CoV-2 epidemic requires intensive testing on the population to early identify and isolate infected subjects. During the first emergency phase of the epidemic, RT-qPCR on nasopharyngeal (NP) swabs, which is the most reliable technique to detect ongoing infections, exhibited limitations due to availability of reagents and budget constraints. This stressed the need to develop screening procedures that require fewer resources and are suitable to be extended to larger portions of the population. RT-qPCR on pooled samples from individual NP swabs seems to be a promising technique to improve surveillance. We performed preliminary experimental analyses aimed to investigate the performance of pool testing on samples with low viral load and we evaluated through Monte Carlo (MC) simulations alternative screening protocols based on sample pooling, tailored to contexts characterized by different infection prevalence. We focused on the role of pool size and the opportunity to develop strategies that take advantage of natural clustering structures in the population, e.g. families, school classes, hospital rooms. Despite the use of a limited number of specimens, our results suggest that, while high viral load samples seem to be detectable even in a pool with 29 negative samples, positive specimens with low viral load may be masked by the negative samples, unless smaller pools are used. The results of MC simulations confirm that pool testing is useful in contexts where the infection prevalence is low. The gain of pool testing in saving resources can be very high, and can be optimized by selecting appropriate group sizes. Exploiting natural groups makes the definition of larger pools convenient and potentially overcomes the issue of low viral load samples by increasing the probability of identifying more than one positive in the same pool.

Registry of patients with multiple sclerosis and COVID-19 infection in Saudi Arabia

BackgroundThe outbreak of coronavirus disease 2019 (COVID-19) has rapidly spread and developed as a pandemic threatening global health. Patients with multiple sclerosis (MS)–an autoimmune demyelinating inflammatory disease of the central nervous system (CNS)–are predominantly treated with immunomodulatory/immunosuppressive disease-modifying therapies (DMTs), which can increase the risk of infection. Therefore, there is concern that these patients may have a higher risk of COVID-19. In response to growing concerns of neurologists and patients, this study aimed to determine the prevalence, severity, and possible complications of COVID-19 infection in patients with MS in Saudi Arabia (SA). MethodsIn this prospective cohort study, demographic and clinical data were obtained from patients residing in SA with MS who had a positive result for COVID-19 per reverse transcription-polymerase chain reaction test or viral gene sequencing, using respiratory or plasma samples. Comparison of COVID-19 severity groups was performed using one-way ANOVA or Kruskal-Wallis test for numerical variables and Chi-squared test for categorical variables. ResultsSeventy patients with MS and COVID-19 (71% female) were included in this analysis. Of the 53 (75.7%) patients receiving a DMT at the time of COVID-19 infection, the most frequently used DMTs were fingolimod (25%) and interferon-beta (25%). Nine (13%) patients had MS relapse and were treated with intravenous methylprednisolone in the four weeks before COVID-19 infection. The most common symptoms at the peak of COVID-19 infection were fever (46%), fatigue (37%), and headache (36%). Symptoms lasted for a mean duration of 8.7 days; all symptomatic patients recovered and no deaths were reported. COVID-19 severity was categorized in three groups: asymptomatic (n = 12), mild–not requiring hospitalization (n = 48), and requiring hospitalization (n = 10; two of whom were admitted to the intensive care unit [ICU]). Between the three groups, comparison of age, body mass index , Expanded Disability Severity Score , MS disease duration, and DMT use at the time of infection showed no significant differences. A higher percentage of patients who were admitted to hospital or the ICU (40%; p = 0.026) presented with an MS relapse within the prior four weeks compared with those who were asymptomatic or had a mild infection (both 8.3%). ConclusionThese findings present a reassuring picture regarding COVID-19 infection in patients with MS. However, patients with MS who have had a relapse in the preceding four weeks (requiring glucocorticoid treatment) may have an increased risk of severe COVID-19.

A Comparative Study of Real-Time RT-PCR–Based SARS-CoV-2 Detection Methods and Its Application to Human-Derived and Surface Swabbed Material

Real-time RT-PCR remains a gold standard in the detection of various viral diseases. In the coronavirus 2019 pandemic, multiple RT-PCR–based tests were developed to screen for viral infection. As an emergency response to increasing testing demand, we established a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PCR diagnostics platform for which we compared different commercial and in-house RT-PCR protocols. Four commercial, one customized, and one in-house RT-PCR protocols were evaluated with 92 SARS-CoV-2–positive and 92 SARS-CoV-2–negative samples. Furthermore, economical and practical characteristics of these protocols were compared. In addition, a highly sensitive digital droplet PCR (ddPCR) method was developed, and application of RT-PCR and ddPCR methods on SARS-CoV-2 environmental samples was examined. Very low limits of detection (1 or 2 viral copies/μL), high sensitivities (93.6% to 97.8%), and high specificities (98.7% to 100%) for the tested RT-PCR protocols were found. Furthermore, the feasibility of downscaling two of the commercial protocols, which could optimize testing capacity, was demonstrated. Tested commercial and customized RT-PCR detection kits show very good and comparable sensitivity and specificity, and the kits could be further optimized for use on SARS-CoV-2 viral samples derived from human and surface swabbed samples.

Phylogenetic Analysis of the 2020 West Nile Virus (WNV) Outbreak in Andalusia (Spain).

During recent decades West Nile Virus (WNV) outbreaks have continuously occurred in the Mediterranean area. In August 2020 a new WNV outbreak affected 71 people with meningoencephalitis in Andalusia and six more cases were detected in Extremadura (south-west of Spain), causing a total of eight deaths. The whole genomes of four viruses were obtained and phylogenetically analyzed in the context of recent outbreaks. The Andalusian viral samples belonged to lineage 1 and were relatively similar to those of previous outbreaks which occurred in the Mediterranean region. Here we present a detailed analysis of the outbreak, including an extensive phylogenetic study. As part on this effort, we implemented a local Nextstrain server, which has become a constituent piece of regional epidemiological surveillance, wherein forthcoming genomes of environmental samples or, eventually, future outbreaks, will be included.

Mass spectrometry-based proteomic platforms for better understanding of SARS-CoV-2 induced pathogenesis and potential diagnostic approaches.

While protein–protein interaction is the first step of the SARS‐CoV‐2 infection, recent comparative proteomic profiling enabled the identification of over 11,000 protein dynamics, thus providing a comprehensive reflection of the molecular mechanisms underlying the cellular system in response to viral infection. Here we summarize and rationalize the results obtained by various mass spectrometry (MS)‐based proteomic approaches applied to the functional characterization of proteins and pathways associated with SARS‐CoV‐2‐mediated infections in humans. Comparative analysis of cell‐lines versus tissue samples indicates that our knowledge in proteome profile alternation in response to SARS‐CoV‐2 infection is still incomplete and the tissue‐specific response to SARS‐CoV‐2 infection can probably not be recapitulated efficiently by in vitro experiments. However, regardless of the viral infection period, sample types, and experimental strategies, a thorough cross‐comparison of the recently published proteome, phosphoproteome, and interactome datasets led to the identification of a common set of proteins and kinases associated with PI3K‐Akt, EGFR, MAPK, Rap1, and AMPK signaling pathways. Ephrin receptor A2 (EPHA2) was identified by 11 studies including all proteomic platforms, suggesting it as a potential future target for SARS‐CoV‐2 infection mechanisms and the development of new therapeutic strategies. We further discuss the potentials of future proteomics strategies for identifying prognostic SARS‐CoV‐2 responsive age‐, gender‐dependent, tissue‐specific protein targets.

Whole Genome Sequencing and Phylogenetic Analysis of SARS‑CoV‑2 strains in Turkey.

Coronaviruses which are single-stranded RNAs, are members of a large family of viruses that may be important pathogens for humans. SARS-CoV-2 was found to cause the severe respiratory syndrome, and on January 22, 2020 first human-to-human transmission was reported. We aimed to reveal the complete genomes of 19 SARS-CoV-2 isolates from Denizli province and identify Turkish patients' genetic similarities. 15 samples with the highest viral loads resulting from RT-PCR were selected for NGS analysis. Fifteen SARS-CoV-2 complete genome sequences were then subjected to phylogenetic analysis and uploaded to the GISAID database. Phylogenetic trees were constructed by the Neighbor-Joining method using MEGAX software. Whole-genome sequencing of the viral RNA samples revealed 32 missense, 21 synonymous, and 4 non-coding alleles. In all samples c.1-25C>T (5'UTR), c.14144C>T (ORF1ab), c.2772C>T (ORF1ab) and c.1841A>G(S) mutations were detected. Phylogenetic analysis revealed that most of the present study's genomes are in 20B clade while the two are in 20A. The phylogenetic tree constructed with all complete SARS-CoV-2 genomes of Turkey showed that the viruses were spread nearly homogenous on eastern (around Kars) and western (around Istanbul) sides. Here, we reported the viral genomes in Denizli comprehensively for the first time. We identified 11 rare missense mutations in the virus compared to the reference genome. Phylogenetic analysis revealed that while most of our isolates were similar to European sequences, some had different sublineages depending on their genomic variants.

The frequency of cytomegalovirus non-ELR UL146 genotypes in neonates with congenital CMV disease is comparable to strains in the background population

BackgroundCongenital cytomegalovirus disease (cCMV) is common and can be fatal or cause severe sequelae. Circulating strains of cytomegalovirus carry a high number of variable or disrupted genes. One of these is UL146, a highly diverse gene with 14 distinct genotypes encoding a CXC-chemokine involved in viral dissemination. UL146 genotypes 5 and 6 lack the conserved ELR motif, potentially affecting strain virulence. Here, we investigate whether UL146 genotypes 5 and 6 were associated with congenital CMV infection.MethodsViral DNA was extracted and UL146 sequenced from 116 neonatal dried blood spots (DBS) stored in the Danish National Biobank since 1982 and linked to registered cCMV cases through a personal identifier. These sequences were compared to UL146 control sequences obtained from CMV DNA extracted from 83 urine samples from children with suspected bacterial urinary tract infections.ResultsThree non-ELR UL146 genotypes (5 and 6) were observed among the cases (2.6%) and two were observed among the controls (2.4%; P > 0.99). Additionally, no significant association with cCMV was found for the other 12 genotypes in a post-hoc analysis, although genotype 8 showed a tendency to be more frequent among cases with 12 observations against three (P = 0.10). All fourteen genotypes were found to have little intra-genotype variation. Viral load, gender, and sample age were not found to be associated with any particular UL146 genotype.ConclusionsNo particular UL146 genotype was associated with cCMV in this nationwide retrospective case-control study. Associations between CMV disease and disrupted or polymorph CMV genes among immunosuppressed people living with HIV/AIDS and transplant recipients should be investigated in future studies.

Profiling SARS-CoV-2 mutation fingerprints that range from the viral pangenome to individual infection quasispecies

BackgroundThe genome of SARS-CoV-2 is susceptible to mutations during viral replication due to the errors generated by RNA-dependent RNA polymerases. These mutations enable the SARS-CoV-2 to evolve into new strains. Viral quasispecies emerge from de novo mutations that occur in individual patients. In combination, these sets of viral mutations provide distinct genetic fingerprints that reveal the patterns of transmission and have utility in contact tracing.MethodsLeveraging thousands of sequenced SARS-CoV-2 genomes, we performed a viral pangenome analysis to identify conserved genomic sequences. We used a rapid and highly efficient computational approach that relies on k-mers, short tracts of sequence, instead of conventional sequence alignment. Using this method, we annotated viral mutation signatures that were associated with specific strains. Based on these highly conserved viral sequences, we developed a rapid and highly scalable targeted sequencing assay to identify mutations, detect quasispecies variants, and identify mutation signatures from patients. These results were compared to the pangenome genetic fingerprints.ResultsWe built a k-mer index for thousands of SARS-CoV-2 genomes and identified conserved genomics regions and landscape of mutations across thousands of virus genomes. We delineated mutation profiles spanning common genetic fingerprints (the combination of mutations in a viral assembly) and a combination of mutations that appear in only a small number of patients. We developed a targeted sequencing assay by selecting primers from the conserved viral genome regions to flank frequent mutations. Using a cohort of 100 SARS-CoV-2 clinical samples, we identified genetic fingerprints consisting of strain-specific mutations seen across populations and de novo quasispecies mutations localized to individual infections. We compared the mutation profiles of viral samples undergoing analysis with the features of the pangenome.ConclusionsWe conducted an analysis for viral mutation profiles that provide the basis of genetic fingerprints. Our study linked pangenome analysis with targeted deep sequenced SARS-CoV-2 clinical samples. We identified quasispecies mutations occurring within individual patients and determined their general prevalence when compared to over 70,000 other strains. Analysis of these genetic fingerprints may provide a way of conducting molecular contact tracing.

Ten scientific reasons in support of airborne transmission of SARS-CoV-2

Heneghan and colleagues' systematic review, funded by WHO, published in March, 2021, as a preprint, states: “The lack of recoverable viral culture samples of SARS-CoV-2 prevents firm conclusions to be drawn about airborne transmission”.1 This conclusion, and the wide circulation of the review's findings, is concerning because of the public health implications.

Detection of group A rotavirus in oyster tissues by in situ capture RT-qPCR

Group A rotavirus (RVA) is the major foodborne pathogen that could be transferred via oysters. There are limitations of current methods for detecting rotavirus in oysters, such as the long duration of the traditional cell culture-based plaque assay, the false positive due to the free RNA using RT-PCR-based molecular methods, and the complex process of RNA extraction from the food matrix. Therefore, we developed a PGM-mediated in situ capture reverse transcription quantitative polymerase chain reaction (ISC-RT-qPCR) assay to investigate the encapsulated RVA in oysters. In parallel comparing tests for the cell-cultured viral samples, the ISC-RT-qPCR exhibited 10-fold greater sensitivity and better accuracy of distinguishing the encapsulated RVA than RT-qPCR. Furthermore, the ISC-RT-qPCR demonstrated a higher detection rate than RT-qPCR when applied to detect RVA in the artificially contaminated oyster. In practical application, the detection rates of RVA in oysters collected from four different farms were respectively higher by ISC-RT-qPCR (37.9%, 28.6%, 27.3%, and 0.0%) than by RT-qPCR (0.0%, 13.8%, 4.5%, and 0.0%), making the ISC-RT-qPCR a better assay for disease monitoring and control. Overall, all results indicate that ISC-RT-qPCR is a promising method for detecting encapsulated RVA in oysters and simplifies the steps of virus concentration, virus extraction with higher sensitivity and accuracy than the conventional RT-qPCR assay.

SARS-CoV-2 presented moderately during two episodes of the infection with lack of antibody responses

The world has gone through the critical phase of SARS-CoV-2 crisis caused by the new variants of the virus. The globally concerted effort to characterize viral genomic mutations across different clades has revealed several changes in the coding and also non-coding regions which might lead to a violent presentation or re-infection occurrence.Here, we studied a COVID-19 subject who represented the symptoms following the full recovery of the first infection. COVID-19 specific IgM and IgG were evaluated in both steps. The viral samples from oropharyngeal/nasopharyngeal were subjected to RT-PCR and full sequencing was done in both incidences. The sequencing data was fully investigated with the reference sequence of SARS-CoV-2 and the changes were detected.The obtained data is in favor of re-infection with 128 days of interval. SARS-CoV-2 presented more severely in the second episode of the disease and the specific antibodies against COVID-19 were not detectable. Both infections were caused by the same clade 20G, however, the mutation rates were higher in the second incidence including 10 nucleotide substitutions which had rarely been reported before. In the present study, the nucleotide mutations in various regions of the viral genome have been presented. The re-infection could have significant effect on clinical implications as well as vaccination.