Virus Concentration Research Articles

Asymmetric Polarization in Online Media Engagement in the United States Congress

In their online communications, political elites may choose to strategically eschew mainstream media sources and engage with alternative media outlets based on ideological and strategic considerations about the type of content they wish to legitimize or amplify to their followers. This is particularly true on social media which has become a fertile breeding ground for viral non-mainstream media content. Using an extensive dataset of social media posts sent from Members of the U.S. Congress between 2011 and 2022 (6.3 million tweets, 2.3 million Facebook posts), this article explores how media engagement in congressional online communications has evolved over time. The results suggest clear trends of asymmetric polarization, both in terms of media link sharing and media handle engagement, with Republican homophily and extremity of engagement growing steadily over time. This growing extremity is explained by changing Member behavior rather than changes in the composition of Congress and is primarily driven by more ideologically extreme Members of the Republican party increasingly engaging with rightwing and alt-right media. These trends have intensified in recent years, especially after the election of Donald Trump in 2016. Collectively, these results shed light on shifting ideological trends in media engagement and have important implications for our understanding of the relationship between political elites and the media in a highly polarized contemporary political environment.

A Robust Phenotypic High-Throughput Antiviral Assay for the Discovery of Rabies Virus Inhibitors.

Rabies virus (RABV) causes severe neurological symptoms in mammals. The disease is almost inevitably lethal as soon as clinical symptoms appear. The use of rabies immunoglobulins (RIG) and vaccination in post-exposure prophylaxis (PEP) can provide efficient protection, but many people do not receive this treatment due to its high cost and/or limited availability. Highly potent small molecule antivirals are urgently needed to treat patients once symptoms develop. In this paper, we report on the development of a high-throughput phenotypic antiviral screening assay based on the infection of BHK-21 cells with a fluorescent reporter virus and high content imaging readout. The assay was used to screen a repurposing library of 3681 drugs (all had been studied in phase 1 clinical trials). From this series, salinomycin was found to selectively inhibit viral replication by blocking infection at the entry stage. This shows that a high-throughput assay enables the screening of large compound libraries for the purposes of identifying inhibitors of RABV replication. These can then be optimized through medicinal chemistry efforts and further developed into urgently needed drugs for the treatment of symptomatic rabies.

Bipolar ionization rapidly inactivates real-world, airborne concentrations of infective respiratory viruses.

The SARS-CoV-2 (COVID-19) pandemic has highlighted the urgent need for strategies that rapidly inactivate airborne respiratory viruses and break the transmission cycle of indoor spaces. Air ions can reduce viable bacteria, mold, and virus counts, however, most studies use small test enclosures with target microbes and ion sources in close vicinity. To evaluate ion performance in real-world spaces, experiments were conducted in a large, room-size BSL-3 Chamber. Negative and positive ions were delivered simultaneously using a commercially available bipolar air ion device. The device housed Needle Point Bipolar ionization (NPBI) technology. Large chamber studies often use unrealistically high virus concentrations to ensure measurable virus is present at the trial end. However, excessively high viral concentrations bias air cleaning devices towards underperformance. Hence, devices that provide a substantial impact for protecting occupants in real-world spaces with real-world virus concentrations are often dismissed as poor performers. Herein, both real-world and excessive virus concentrations were studied using Influenza A and B, Human Respiratory Syncytial Virus (RSV), and the SARS-CoV-2 Alpha and Delta strains. The average ion concentrations ranged from 4,100 to 24,000 per polarity over 60-minute and 30-minute time trials. The reduction rate was considerably greater for trials that used real-world virus concentrations, reducing infectivity for Influenza A and B, RSV, and SARS-CoV-2 Delta by 88.3-99.98% in 30 minutes, whereas trials using in-excess concentrations showed 49.5-61.2% in 30 minutes. These findings strongly support the addition of NPBI ion technology to building management strategies aimed to protect occupants from contracting and spreading infective respiratory viruses indoors.

Diurnal changes in pathogenic and indicator virus concentrations in wastewater

Wastewater-based epidemiology (WBE) has been commonly used for monitoring SARS-CoV-2 outbreaks. As sampling times and methods (i.e. grab vs composite) may vary, diurnal changes of viral concentrations in sewage should be better understood. In this study, we collected untreated wastewater samples hourly for 4 days at two wastewater treatment plants in Wales to establish diurnal patterns in virus concentrations and the physico-chemical properties of the water. Simultaneously, we also trialled three absorbent materials as passive samples as a simple and cost-efficient alternative for the collection of composite samples. Ninety-six percent of all liquid samples (n = 74) and 88% of the passive samplers (n = 59) were positive for SARS-CoV-2, whereas 87% and 97% of the liquid and passive samples were positive for the faecal indicator virus crAssphage, respectively. We found no significant daily variations in the concentration of the target viruses, ammonium and orthophosphate, and the pH and electrical conductivity levels were also stable. Weak positive correlations were found between some physico-chemical properties and viral concentrations. More variation was observed in samples taken from the influent stream as opposed to those taken from the influent tank. Of the absorbent materials trialled as passive samples, we found that tampons provided higher viral recoveries than electronegative filter paper and cotton gauze swabs. For all materials tested, viral recovery was dependent on the virus type. Our results indicate that grab samples may provide representative alternatives to 24-h composite samples if taken from the influent tank, hence reducing the costs of sampling for WBE programmes. Tampons are also viable alternatives for cost-efficient sampling; however, viral recovery should be optimised prior to use.

Improvement of the qmosRT-PCR Assay and Its Application for the Detection and Quantitation of the Three Serotypes of the Novel Oral Polio Vaccine in Stool Samples.

Recently, genetically stable novel OPVs (nOPV) were developed by modifying the genomes of Sabin viruses of conventional OPVs to reduce the risk of reversion to neurovirulence and therefore the risk of generating circulating vaccine-derived polioviruses. There is a need for specific and sensitive methods for the identification and quantification of nOPV viruses individually and in mixtures for clinical trials and potentially for manufacturing quality control and environmental surveillance. In this communication, we evaluated and improved the quantitative multiplex one-step reverse transcriptase polymerase chain reaction (qmosRT-PCR) assay for the identification and quantification of nOPV viruses in samples with different formulations and virus concentrations and in virus-spiked stool samples. The assay was able to specifically identify at least 1 log10 CCID50/mL of each serotype in the presence of the two other serotypes at high concentrations (6-7 log10 CCID50/mL) in the same sample. In addition, the lowest viral concentration that the assay was able to detect in stool samples was 17 CCID50/mL for nOPV1 and nOPV2 viruses and 6 CCID50/mL for nOPV3. We also found high correlation between the expected and observed (by qmosRT-PCR) concentrations of spiked viruses in stool samples for all three nOPV viruses, with R-squared values above 0.95. The analysis of samples collected from an nOPV2 clinical trial showed that 100% of poliovirus type 2 was detected and few samples showed the presence of type 1 and 3 residuals from previous vaccinations with bOPV (at least 4 weeks prior vaccination with nOPV2), confirming the high sensitivity of the method. The qmosRT-PCR was specific and sensitive for the simultaneous identification and quantification of all three nOPV viruses. It can be used as an identity test during the nOPV manufacturing process and in evaluation of virus excretion in nOPV clinical trials.

Performance of SARS-CoV-2 nucleic acid amplification testing in Austria as measured by external quality assessment schemes during 3 years of the COVID-19 pandemic: an observational retrospective study

The aim of external quality assessment (EQA) schemes is to evaluate the analytical performance of laboratories and test systems in a near-to-real-life setting. This monitoring service provides feedback to participant laboratories and serves as a control measure for the epidemiological assessment of the regional incidence of a pathogen, particularly during epidemics. Using data from EQA schemes implemented as a result of the intensive effort to monitor SARS-CoV-2 infections in Austria, we aimed to identify factors that explained the variation in laboratory performance for SARS-CoV-2 detection over the course of the COVID-19 pandemic. For this observational study, we retrospectively analysed 6308 reverse transcriptase quantitative PCR (RT-qPCR) test results reported by 191 laboratories on 71 samples during 14 rounds of three SARS-CoV-2 pathogen detection EQA schemes in Austria between May 18, 2020, and Feb 20, 2023. We calculated the overall rates of false and true-negative, false and true-positive, and inconclusive results. We then assessed laboratory performance by estimating the sensitivity by testing whether significant variation in the odds of obtaining a true-positive result could be explained by virus concentration, laboratory type, or assay format. We also assessed whether laboratory performance changed over time. 4371 (93·7%) of 4663 qPCR test results were true-positive, 241 (5·2%) were false-negative, and 51 (1·1%) were inconclusive. The mean per-sample sensitivity was 99·7% in samples with high virus concentrations (1383 [99·4%] true-positive, three [0·2%] false-negative, and five [0·4%] inconclusive results for 1391 tests in which the sample cycle threshold was ≤32), whereas detection rates were lower in samples with low virus concentrations (mean per-sample sensitivity 92·5%; 2988 [91·3%] true-positive, 238 [7·3%] false-negative, and 46 [1·4%] inconclusive results for 3272 tests in which the cycle threshold was >32). Of the 1645 results expected to be negative, 1561 (94·9%) were correctly reported as negative, 10 (0·6%) were incorrectly reported as positive, and 74 (4·5%) were reported as inconclusive. Notably, the overall performance of the tests did not change significantly over time. The odds of reporting a correct result were 2·94 (95% CI 1·75-4·96) times higher for a medical laboratory than for a non-medical laboratory, and 4·60 (2·91-7·41) times greater for automated test systems than for manual test systems. Automated test systems within medical laboratories had the highest sensitivity when compared with systems requiring manual intervention in both medical and non-medical laboratories. High rates of false-negativity in all PCR analyses evaluated in comprehensive, multiple, and repeated EQA schemes outline a clear path for improvement in the future. The performance of some laboratories (eg, non-medical laboratories or those using non-automated test systems) should receive additional scrutiny-for example, by requiring additional EQA schemes for certification or accreditation-if the aggregated data from EQA rounds suggest lower sensitivity than that recorded by others. This strategy will provide assurances that epidemiological data as a whole are reliable when testing on such a large scale. Although performance did not improve over time, we cannot exclude extenuating circumstances-such as shortages and weakened supply chains-that could have prevented laboratories from seeking alternative methods to improve performance. None.

Mathematical analysis of global dynamics and optimal control of treatment for an age-structured HBV infection model

Evidences show that cell-to-cell infection occurs in HBV infection and is a very efficient way which may lead to more than half of the viral infection. Hence we formulate an HBV infection model with cell-to-cell infection, meanwhile, we incorporate the infection age of infected cells. For dynamic behaviors, we firstly establish the well-posedness and asymptotic smoothness of the model; then show the global threshold dynamical behaviors, which are determined by the reproduction number; and finally perform numerical simulations not only to verify the theoretical results but also to explore effects of cell-to-cell infection on viral load and infection concentration. Our finding is that ignoring the cell-to-cell infection will underestimate the actual HBV infection status. For optimal control of treatment for HBV, we introduce two time-varying control measures and investigate the corresponding optimal control problem. We derive the characteristic expression of the optimal control and discuss effects of control costs and integrated control. The results show that high control cost will not result in better control effectiveness, while integrated optimal control can reach the best control objectives compared with the two single ones. Moreover, infection age can greatly influence HBV infection at the initial stage.

Metatranscriptome of human lung microbial communities in a cohort of mechanically ventilated COVID-19 Omicron patients

The Omicron variant of the severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) infected a substantial proportion of Chinese population, and understanding the factors underlying the severity of the disease and fatality is valuable for future prevention and clinical treatment. We recruited 64 patients with invasive ventilation for COVID-19 and performed metatranscriptomic sequencing to profile host transcriptomic profiles, plus viral, bacterial, and fungal content, as well as virulence factors and examined their relationships to 28-day mortality were examined. In addition, the bronchoalveolar lavage fluid (BALF) samples from invasive ventilated hospital/community-acquired pneumonia patients (HAP/CAP) sampled in 2019 were included for comparison. Genomic analysis revealed that all Omicron strains belong to BA.5 and BF.7 sub-lineages, with no difference in 28-day mortality between them. Compared to HAP/CAP cohort, invasive ventilated COVID-19 patients have distinct host transcriptomic and microbial signatures in the lower respiratory tract; and in the COVID-19 non-survivors, we found significantly lower gene expressions in pathways related viral processes and positive regulation of protein localization to plasma membrane, higher abundance of opportunistic pathogens including bacterial Alloprevotella, Caulobacter, Escherichia-Shigella, Ralstonia and fungal Aspergillus sydowii and Penicillium rubens. Correlational analysis further revealed significant associations between host immune responses and microbial compositions, besides synergy within viral, bacterial, and fungal pathogens. Our study presents the relationships of lower respiratory tract microbiome and transcriptome in invasive ventilated COVID-19 patients, providing the basis for future clinical treatment and reduction of fatality.

A COVID-19 cluster analysis in an office: Assessing the long-range aerosol and fomite transmissions with infection control measures.

Simulated exposure to severe acute respiratory syndrome coronavirus 2 in the environment was demonstrated based on the actual coronavirus disease 2019 cluster occurrence in an office, with a projected risk considering the likely transmission pathways via aerosols and fomites. A total of 35/85 occupants were infected, with the attack rate in the first stage as 0.30. It was inferred that the aerosol transmission at long-range produced the cluster at virus concentration in the saliva of the infected cases on the basis of the simulation, more than 108PFUmL-1. Additionally, all wearing masks effectiveness was estimated to be 61%-81% and 88%-95% reduction in risk for long-range aerosol transmission in the normal and fit state of the masks, respectively, and a 99.8% or above decline in risk of fomite transmission. The ventilation effectiveness for long-range aerosol transmission was also calculated to be 12%-29% and 36%-66% reductions with increases from one air change per hour (ACH) to two ACH and six ACH, respectively. Furthermore, the virus concentration reduction in the saliva to 1/3 corresponded to the risk reduction for long-range aerosol transmission by 60%-64% and 40%-51% with and without masks, respectively.

Depth-driven patterns in lytic viral diversity, auxiliary metabolic gene content, and productivity in offshore oligotrophic waters.

Marine viruses regulate microbial population dynamics and biogeochemical cycling in the oceans. The ability of viruses to manipulate hosts' metabolism through the expression of viral auxiliary metabolic genes (AMGs) was recently highlighted, having important implications in energy production and flow in various aquatic environments. Up to now, the presence and diversity of viral AMGs is studied using -omics data, and rarely using quantitative measures of viral activity alongside. In the present study, four depth layers (5, 50, 75, and 1,000 m) with discrete hydrographic features were sampled in the Eastern Mediterranean Sea; we studied lytic viral community composition and AMG content through metagenomics, and lytic production rates through the viral reduction approach in the ultra-oligotrophic Levantine basin where knowledge regarding viral actions is rather limited. Our results demonstrate depth-dependent patterns in viral diversity and AMG content, related to differences in temperature, nutrients availability, and host bacterial productivity and abundance. Although lytic viral production rates were similar along the water column, the virus-to-bacteria ratio was higher and the particular set of AMGs was more diverse in the bathypelagic (1,000 m) than the shallow epipelagic (5, 50, and 75 m) layers, revealing that the quantitative effect of viruses on their hosts may be the same along the water column through the intervention of different AMGs. In the resource- and energy-limited bathypelagic waters of the Eastern Mediterranean, the detected AMGs could divert hosts' metabolism toward energy production, through a boost in gluconeogenesis, fatty-acid and glycan biosynthesis and metabolism, and sulfur relay. Near the deep-chlorophyll maximum depth, an exceptionally high percentage of AMGs related to photosynthesis was noticed. Taken together our findings suggest that the roles of viruses in the deep sea might be even more important than previously thought as they seem to orchestrate energy acquisition and microbial community dynamics, and thus, biogeochemical turnover in the oceans.

PCR standard curve quantification in an extensive wastewater surveillance program: results from the Dutch SARS-CoV-2 wastewater surveillance.

Since the start of the COVID-19 pandemic in 2020, wastewater surveillance programs were established, or upscaled, in many countries around the world and have proven to be a cost-effective way of monitoring infectious disease pathogens. Many of these programs use RT-qPCR, and quantify the viral concentrations in samples based on standard curves, by including preparations of a reference material with known nucleic acid or virus concentrations in the RT-qPCR analyses. In high-throughput monitoring programs it is possible to combine data from multiple previous runs, circumventing the need for duplication and resulting in decreased costs and prolonged periods during which the reference material is obtained from the same batch. However, over time, systematic shifts in standard curves are likely to occur. This would affect the reliability and usefulness of wastewater surveillance as a whole. We aim to find an optimal combination of standard curve data to compensate for run-to-run measurement variance while ensuring enough flexibility to capture systematic longitudinal shifts. Based on more than 4000 observations obtained with the CDC N1 and N2 assays, taken as a part of the National Sewage Surveillance program at the Dutch National Institute for Public Health and the Environment, we show that seasonal and long-term shifts in RT-qPCR efficiency and sensitivity occur. We find that in our setting, using five days of standard-curve data to quantify, results in the least error prone curve or best approximation. This results in differences up to 100% in quantified viral loads when averaged out over a nationwide program of >300 treatment plants. Results show that combining standard curves from a limited set of runs can be a valid approach to quantification without obscuring the trends in the viral load of interest.

Numerical simulation study of indoor disinfection spray distribution based on CFD-DPM method

In today's world, environmental safety has become a top priority, with a special focus on terminal disinfection of indoor environments, which has proven to be a crucial and formidable issue in infection control. The use of hydrogen peroxide disinfection through vaporization or atomization presents a viable solution to the challenges posed by traditional wiping disinfection and ultraviolet disinfection methods that are plagued by incomplete disinfection and other factors that can significantly reduce the virus content within a room. To improve the application of hydrogen peroxide disinfection spray, the CFD-DPM (Computational Fluid Dynamics-Discrete Particle Model) method is employed to simulate the particle distribution of traditional sprays in a 30 m3 room. The study places emphasis on analyzing the impact of the placement position and bottle number of disinfection spray on the distribution of spray particles. The results demonstrate an impressive consistency between the numerical simulation method and experimental findings regarding the particle size distribution of disinfectant spray across various Schemes, validating the method's accuracy and reliability. However, the placement of a single disinfection spray source is insufficient to achieve uniformly distributed particles throughout the space when a bathroom is present within a given room. Nevertheless, by placing internal and external sources within the bathroom, the distribution of disinfectant spray particles becomes more even, thanks to a well conceived placement technique and an increased number of spray sources. Among all the schemes tested, Scheme 4 stands out for its ability to produce the greatest number of spray particles, with almost 60.71% of these particles remaining airborne for over 40 s. Therefore, it is also the most effective scheme for conducting disinfection.

Fig Mosaic Disease (FMD) and Extracts by Water (Aqueous Extracts)

There is a lot of research on the topic of control on Fig mosaic disease (FMD), aim was to reduce damage it causes to fig trees or seedlings. in this study was controlled on fig infected seedlings by aqueous extracts and the results of the treatments in the open field for the cultivation of fig seedlings achieved significant differences in the severity of infection and growth parameters. It gave overlap treatment to ginger roots and mint leaves extract severity injury lowest by virus and highest content of chlorophyll, leaf area, number of leaves and height to infected seedlings reached 22.34%,73.37 mg / 100 gm1- moist weight, 22.48 cm,22.57 leaf/plant,36.73 cm respectively compared with control treatments that achieved 91.18%,47.30 mg / 100 gm1- moist weight, 3.61 cm,8.33 leaf/plant,17.57 cm respectively.

Prokaryotic Plankton and Viruses in the Waters of the Fram Strait in the Winter Period

New data have been obtained on the distribution of prokaryotic plankton and pelagic viruses in the Fram Strait (the Greenland Sea) during the polar night (late November). Three main types of water masses were present in the study area: surface polar, Atlantic, and transformed Atlantic. The content of mineral biogenic elements increased with depth. The concentration of chlorophyll a had low values (0.07–0.13 mg/m3). The abundance and biomass of prokaryotes varied from 286 000 to 675 000 cells/mL and from 2.7 to 11.7 mg C/m3, respectively. The composition of prokaryotic plankton was dominated by single small cells (more than 97% of the abundance and more than 68% of the biomass). The average cell volume was 0.034–0.096 µm3. The number of viruses varied from 724 000 to 3 920 000 particles/mL, and the biomass varied from 0.040 to 0.216 μg C/m3. Against the background of local maxima in the abundance of prokaryotic plankton in the 0–25 m layer, the highest concentrations of viruses were noted in the surface layer. In the latitudinal distribution (from south to north), an increase in the number of viruses and prokaryotes was revealed. A close relationship between their abundance and biomass with hydrological parameters and phosphate content was established; the role of certain water masses in the vertical distribution of microbes was insignificant. The relatively high abundance of viruses and prokaryotes indicated their significant activity during the polar night.

Metabarcoding of Hepatitis E Virus Genotype 3 and Norovirus GII from Wastewater Samples in England Using Nanopore Sequencing

Norovirus is one of the largest causes of gastroenteritis worldwide, and Hepatitis E virus (HEV) is an emerging pathogen that has become the most dominant cause of acute viral hepatitis in recent years. The presence of norovirus and HEV has been reported within wastewater in many countries previously. Here we used amplicon deep sequencing (metabarcoding) to identify norovirus and HEV strains in wastewater samples from England collected in 2019 and 2020. For HEV, we sequenced a fragment of the RNA-dependent RNA polymerase (RdRp) gene targeting genotype three strains. For norovirus, we sequenced the 5′ portion of the major capsid protein gene (VP1) of genogroup II strains. Sequencing of the wastewater samples revealed eight different genotypes of norovirus GII (GII.2, GII.3, GII.4, GII.6, GII.7, GII.9, GII.13 and GII.17). Genotypes GII.3 and GII.4 were the most commonly found. The HEV metabarcoding assay was able to identify HEV genotype 3 strains in some samples with a very low viral concentration determined by RT-qPCR. Analysis showed that most HEV strains found in influent wastewater were typed as G3c and G3e and were likely to have originated from humans or swine. However, the small size of the HEV nested PCR amplicon could cause issues with typing, and so this method is more appropriate for samples with high CTs where methods targeting longer genomic regions are unlikely to be successful. This is the first report of HEV RNA in wastewater in England. This study demonstrates the utility of wastewater sequencing and the need for wider surveillance of norovirus and HEV within host species and environments.

Effect of wastewater collection and concentration methods on assessment of viral diversity

Monitoring of potentially pathogenic human viruses in wastewater is of crucial importance to understand disease trends in communities, predict potential outbreaks, and boost preparedness and response by public health departments. High throughput metagenomic sequencing opens an opportunity to expand the capabilities of wastewater surveillance. However, there are major bottlenecks in the metagenomic enabled wastewater surveillance, including the complexities in selecting appropriate sampling and concentration/virus enrichment methods as well as in bioinformatic analysis of complex samples with low human virus concentrations. To evaluate the abilities of two commonly used sampling and concentration methods in virus identification, virus communities concentrated with Virus Adsorption-Elution (VIRADEL) and PolyEthylene Glycol (PEG) precipitation were compared for three interceptor sites. Results indicated that more viral reads were obtained by the VIRADEL concentration method, with 2.84 ± 0.57 % viral reads in the sample. For samples concentrated with PEG, the average proportion of viral reads in the sample was 0.63 ± 0.19 %. In all wastewater samples, bacteriophage affiliated with the families Siphoviridae, Myoviridae and Podoviridae were found to be the abundant populations. Comparison against a custom Swiss-Prot human virus database indicated that the relatively abundant human viruses (average proportions in human virus community greater than 1.00 %) in samples concentrated with the VIRADEL method were Orthopoxvirus, Rhadinovirus, Parapoxvirus, Varicellovirus, Hepatovirus, Simplexvirus, Molluscipoxvirus, Parechovirus, Lymphocryptovirus, and Spumavirus. In samples concentrated with the PEG method, fewer human viruses were found to be relatively abundant. These were Orthopoxvirus, Rhadinovirus, Varicellovirus, Simplexvirus, Molluscipoxvirus, Lymphocryptovirus, and Betacoronavirus. Contigs of Betacoronavirus, which contains severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), were identified in VIRADEL and PEG samples. Our study demonstrates the feasibility of using metagenomics in wastewater surveillance as a first screening tool and the need for selecting the appropriate virus concentration methods and optimizing bioinformatic approaches in analyzing metagenomic data of wastewater samples.

Universal primers for rift valley fever virus whole-genome sequencing

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease causing acute hemorrhagic fever. Accurate identification of mutations and phylogenetic characterization of RVF virus (RVFV) require whole-genome analysis. Universal primers to amplify the entire RVFV genome from clinical samples with low copy numbers are currently unavailable. Thus, we aimed to develop universal primers applicable for all known RVFV strains. Based on the genome sequences available from public databases, we designed eight pairs of universal PCR primers covering the entire RVFV genome. To evaluate primer universality, four RVFV strains (ZH548, Kenya 56 (IB8), BIME-01, and Lunyo), encompassing viral phylogenetic diversity, were chosen. The nucleic acids of the test strains were chemically synthesized or extracted via cell culture. These RNAs were evaluated using the PCR primers, resulting in successful amplification with expected sizes (0.8–1.7 kb). Sequencing confirmed that the products covered the entire genome of the RVFV strains tested. Primer specificity was confirmed via in silico comparison against all non-redundant nucleotide sequences using the BLASTn alignment tool in the NCBI database. To assess the clinical applicability of the primers, mock clinical specimens containing human and RVFV RNAs were prepared. The entire RVFV genome was successfully amplified and sequenced at a viral concentration of 108 copies/mL. Given the universality, specificity, and clinical applicability of the primers, we anticipate that the RVFV universal primer pairs and the developed method will aid in RVFV phylogenomics and mutation detection.

Development of potentiometric immunosensor for determination of live attenuated Varicella Vaccine: Potency and stability studies

Determination of Varicella vaccine's potency; containing live attenuated strain (Oka) of Varicella Zoster virus, has been limited to in vitro cell culture methods. In this study, a label free potentiometric biosensor has been developed for the first time and optimized to determine the content of varicella zoster virus. A passive ion-flux sensing platform has been developed using an anti-varicella monoclonal antibody and tetrabutyl ammonium bromide as a marker ion. The immunosensor has been optimized with respect to membrane diameter and concentration of the immobilized antibody. Linearity was achieved over a concentration range of 2.5–3.2 log PFU/dose with a LOD of 1.9 log PFU/dose. Potentiometric results were compared to the plaque-forming assay using the cell culture technique. The developed immunosensor was superior with respect to analysis time and cost without affecting critical analytical performance characteristics. Furthermore, in order to evaluate the stability indicating ability of the immunosensor, the effect of pH and temperature was investigated. Vaccine samples were subjected to forced degradation conditions; pH and elevated temperatures. Stability results showed the ability of immunosensor to differentiate between intact and degraded viral content. This would demonstrate the reliability of the immunosensor for evaluating the efficacy and stability of the vaccine.

Comparison of adsorption-extraction (AE) workflows for improved measurements of viral and bacterial nucleic acid in untreated wastewater

The lack of standardized methods and large differences in virus concentration and extraction workflows have hampered Severe Acute Respiratory Syndrome (SARS-CoV-2) wastewater surveillance and data reporting practices. Numerous studies have shown that adsorption-extraction (AE) method holds promise, yet several uncertainties remain regarding the optimal AE workflow. Several procedural components may influence the recovered concentrations of target nucleic acid, including membrane types, homogenization instruments, speed and duration, and lysis buffer. In this study, 42 different AE workflows that varied these components were compared to determine the optimal workflow by quantifying endogenous SARS-CoV-2, human adenovirus 40/41 (HAdV 40/41), and a bacterial marker gene of fecal contamination (Bacteroides HF183). Our findings suggest that the workflow chosen had a significant impact on SARS-CoV-2 concentrations, whereas it had minimal impact on HF183 and no effect on HAdV 40/41 concentrations. When comparing individual components in a workflow, such as membrane type (MF-Millipore™ 0.45 μm MCE vs. Isopore™ 0.40 μm), we found that they had no impact on SARS-CoV-2, HAdV 40/41, and HF183 concentrations. This suggests that at least some consumables and equipment are interchangeable. Buffer PM1 + TRIzol-based workflows yielded higher concentrations of SARS-CoV-2 than other workflows. HF183 concentrations were higher in workflows without chloroform. Similarly, higher homogenization speeds (5000–10,000 rpm) led to increased concentrations of SARS-CoV-2 and HF183 but had no effect on HAdV 40/41. Our findings indicate that minor enhancements to the AE workflow can improve the recovery of viruses and bacteria from the wastewater, leading to improved outcomes from wastewater surveillance efforts.

Comparison of metagenomic and targeted methods for sequencing human pathogenic viruses from wastewater.

Most public health initiatives that monitor viruses in wastewater have utilized quantitative polymerase chain reaction (PCR) and whole genome PCR sequencing, mirroring techniques used for viral epidemiology in individuals. These techniques require prior knowledge of the target viral genome and are limited to monitoring individual or small groups of viruses. Metagenomic sequencing may offer an alternative strategy for monitoring a broad spectrum of viruses in wastewater, including novel and emerging pathogens. In this study, while amplicon sequencing gave high viral genome coverage, untargeted shotgun sequencing of total nucleic acid samples was unable to detect human pathogenic viruses with enough sensitivity for use in genomic epidemiology. Enrichment of shotgun libraries for respiratory viruses using hybrid-capture technology provided genotypic information on a range of viruses simultaneously, indicating strong potential for wastewater surveillance. This type of targeted metagenomics could be used for monitoring diverse targets, such as pathogens or antimicrobial resistance genes, in environmental samples.