Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Development of a robust cell-based potency assay for a coxsackievirus A21 oncolytic virotherapy.

Oncolytic viruses (OV) are part of a burgeoning field of investigational oncolytic therapy (OT), in which lytic viruses dissolve advanced tumors productively and specifically. One such OT is a Coxsackievirus A21 (CVA21) based OV that is currently under clinical evaluation. A tissue culture infectious dose (TCID50) assay was used for CVA21 potency release and stability testing in early clinical development. The titer measured in this method was an extrapolated value from cytopathic effect (CPE) observed during the serial dilution but doesn't represent direct viral killing of cells. Moreover, the assay was not deemed to be optimal to carry into late phase clinical development due to limitations in assay precision, turn-around time, and sample throughput. To address these points, we developed a plaque assay to measure viral plaque forming units to measure the potency value for drug substance (DS), drug product (DP) and virus seed (master and working) stocks. In this manuscript, we describe the steps taken to develop this plaque assay for the late-stage clinical development, which include the assay qualification, validation, and robustness protocols, and describe statistical methods for data analysis. Moreover, the method was validated for linearity, accuracy, precision, and specificity. Furthermore, the plaque assay quantifies OV infectivity with better precision (32% vs 58%), with higher sample throughput (22 samples/week vs 3 samples/week) and shorter assay turnaround time (4 days vs 7 days) than the TCID50 method. This assay development strategy can provide guidance for the development of robust cell-based potency methods for OVs and other infectious viral products.

Development and Validation of a Plaque Assay to Determine the Titer of a Recombinant Live-Attenuated Viral Vaccine for SARS-CoV-2.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in more than seven million deaths worldwide. To reduce viral spread, the Israel Institute for Biological Research (IIBR) developed and produced a new rVSV-SARS-CoV-2-S vaccine candidate (BriLife®) based on a platform of a genetically engineered vesicular stomatitis virus (VSV) vector that expresses the spike protein of SARS-CoV-2 instead of the VSV-G protein on the virus surface. Quantifying the virus titer to evaluate vaccine potency requires a reliable validated assay that meets all the stringent pharmacopeial requirements of a bioanalytical method. Here, for the first time, we present the development and extensive validation of a quantitative plaque assay using Vero E6 cells for the determination of the concentration of the rVSV-SARS-CoV-2-S viral vector. Three different vaccine preparations with varying titers (DP_low, DP_high, and QC sample) were tested according to a strict validation protocol. The newly developed plaque assay was found to be highly specific, accurate, precise, and robust. The mean deviations from the predetermined titers for the DP_low, DP_high, and QC preparations were 0.01, 0.02, and 0.09 log10, respectively. Moreover, the mean %CV values for intra-assay precision were 18.7%, 12.0%, and 6.0%, respectively. The virus titers did not deviate from the established values between cell passages 5 and 19, and no correlation was found between titer and passage. The validation results presented herein indicate that the newly developed plaque assay can be used to determine the concentration of the BriLife® vaccine, suggesting that the current protocol is a reliable methodology for validating plaque assays for other viral vaccines.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

Vector competence of Swedish Culex pipiens mosquitoes for Usutu virus.

Usutu virus (USUV) is an emerging mosquito-borne flavivirus with increasing prevalence in Europe. Understanding the role of mosquito species in USUV transmission is crucial for predicting and controlling potential outbreaks. This study aimed to assess the vector competence of Swedish Culex pipiens for USUV. The mosquitoes were orally infected with an Italian strain of USUV (Bologna 2009) and infection rates (IR), dissemination rates (DR), and transmission rates (TR) were evaluated over 7 to 28 days post-infection. The study revealed that Swedish Cx. pipiens are susceptible to USUV infection, with a gradual decrease in IR over time. However, the percentage of mosquitoes with the ability to transmit the virus remained consistent across all time points, indicating a relatively short extrinsic incubation period. Overall, this research highlights the potential of Swedish Cx. pipiens as vectors for USUV and emphasizes the importance of surveillance and monitoring to prevent future outbreaks of mosquito-borne diseases.

Culturing of SARS-CoV-2 from patient samples: Protocol for optimal virus recovery and assessment of infectious viral load.

Optimal sampling, preservation, and culturing of SARS-CoV-2 from COVID-19 patients are critical for successful recovery of virus isolates and to accurately estimate contagiousness of the patient. In this study, we investigated the influence of the type of sampling media, storage time, freezing conditions, sterile filtration, and combinations of these to determine the optimal pre-analytic conditions for virus recovery and estimation of infectious viral load in COVID-19 patients. Further, we investigated the viral shedding kinetics and mucosal antibody response in 38 COVID-19 hospitalized patients. We found Universal Transport Medium (Copan) to be the most optimal medium for preservation of SARS-CoV-2 infectivity. Our data showed that the probability of a positive viral culture was strongly correlated to Ct values, however some samples did not follow the general trend. We found a significant correlation between plaque forming units and levels of mucosal antibodies and found that high levels of mucosal antibodies correlated with reduced chance of isolating the virus. Our data reveals essential parameters to consider from specimen collection over storage to culturing technique for optimal chance of isolating SARS-CoV-2 and accurately estimating patient contagiousness.

Cryptic bacterial pathogens of diatoms peak during senescence of a winter diatom bloom.

Diatoms are globally abundant microalgae that form extensive blooms in aquatic ecosystems. Certain bacteria behave antagonistically towards diatoms, killing or inhibiting their growth. Despite their crucial implications to diatom blooms and population health, knowledge of diatom antagonists in the environment is fundamentally lacking. We report systematic characterisation of the diversity and seasonal dynamics of bacterial antagonists of diatoms via plaque assay sampling in the Western English Channel (WEC), where diatoms frequently bloom. Unexpectedly, peaks in detection did not occur during characteristic spring diatom blooms, but coincided with a winter bloom of Coscinodiscus, suggesting that these bacteria likely influence distinct diatom host populations. We isolated multiple bacterial antagonists, spanning 4 classes and 10 bacterial orders. Notably, a diatom attaching Roseobacter Ponticoccus alexandrii was isolated multiple times, indicative of a persistent environmental presence. Moreover, many isolates had no prior reports of antagonistic activity towards diatoms. We verified diatom growth inhibitory effects of eight isolates. In all cases tested, these effects were activated by pre-exposure to diatom organic matter. Discovery of widespread 'cryptic' antagonistic activity indicates that bacterial pathogenicity towards diatoms is more prevalent than previously recognised. Finally, examination of the global biogeography of WEC antagonists revealed co-occurrence patterns with diatom host populations in marine waters globally.

Reverse Genetics System for Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important mosquito-borne virus that can cause severe disease manifestations in humans including ocular damage, vision loss, late-onset encephalitis, and hemorrhagic fever. In ruminants, RVFV can cause high mortality rates in young animals and high rates of abortion in pregnant animals resulting in an enormous negative impact on the economy of affected regions. To date, no licensed vaccines in humans or anti-RVFV therapeutics for animal or human use are available. The development of reverse genetics has facilitated the generation of recombinant infectious viruses that serve as powerful tools for investigating the molecular biology and pathogenesis of RVFV. Infectious recombinant RVFV can be rescued entirely from cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of pathogenesis and generate live-attenuated vaccines. In this chapter, we will describe the experimental procedures for the implementation of RVFV reverse genetics.

The Virus-Induced Cytopathic Effect.

The cytopathic effect comprises the set of cellular alterations produced by a viral infection. It is of great relevance since it constitutes a direct marker of infection. Likewise, these alterations are often virus-specific which makes them a phenotypic marker for many viral species. All these characteristics have been used to complement the study of the dynamics of virus-cell interactions through the kinetic study of the progression of damage produced by the infection. Various approaches have been used to monitor the cytopathic effect, ranging from light microscopy, immunofluorescence assays, and direct labeling with fluorescent dyes, to plaque assay for the characterization of the infection over time. Here we address the relevance of the study of cytopathic effect and describe different experimental alternatives for its application.

Water-in-oil droplet-mediated method for detecting and isolating infectious bacteriophage particles via fluorescent staining.

Bacteriophages are the most abundant entities on Earth. In contrast with the number of phages considered to be in existence, current phage isolation and screening methods lack throughput. Droplet microfluidic technology has been established as a platform for high-throughput screening of biological and biochemical components. In this study, we developed a proof-of-concept method for isolating phages using water-in-oil droplets (droplets) as individual chambers for phage propagation and co-cultivating T2 phage and their host cell Escherichia coli within droplets. Liquid cultivation of microbes will facilitate the use of microbes that cannot grow on or degrade agar as host cells, ultimately resulting in the acquisition of phages that infect less known bacterial cells. The compartmentalizing characteristic of droplets and the use of a fluorescent dye to stain phages simultaneously enabled the enumeration and isolation of viable phage particles. We successfully recultivated the phages after simultaneously segregating single phage particles into droplets and inoculating them with their host cells within droplets. By recovering individual droplets into 96-well plates, we were able to isolate phage clones derived from single phage particles. The success rate for phage recovery was 35.7%. This study lays the building foundations for techniques yet to be developed that will involve the isolation and rupturing of droplets and provides a robust method for phage enumeration and isolation.

Interlaboratory comparison of Pseudomonas aeruginosa phage susceptibility testing.

Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P. aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP8), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P. aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Improved Culture Methods for Human Coronaviruses HCoV-OC43, HCoV-229E, and HCoV-NL63.

HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 are four of the seven known human coronaviruses (HCoVs) and, unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, these four so-called seasonal HCoVs generally cause mild upper-respiratory-tract illness. As Biosafety Level 2 (BSL-2) pathogens, the seasonal HCoVs are more accessible and can be used as surrogates for studying the highly pathogenic HCoVs. However, scientists have for many years found these difficult to study because of the lack of a universal culture system and the inability of typical culture methods to yield high-titer infectious stocks. We have developed assays to grow and quantify infectious virus and viral RNA for HCoV-OC43, -229E, and -NL63. We identified which immortalized cell lines should be used to optimize the replication of HCoV-OC43, -229E, and -NL63 in order to generate high titers (Vero E6, Huh-7, and LLC-MK2 cells, respectively). Here we present protocols for improved propagation and quantification of each seasonal HCoV. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Growth of HCoVs Basic Protocol 2: Quantification of HCoV by plaque assay Basic Protocol 3: Quantification of HCoV RNA products of replication Basic Protocol 4: Concentrating HCoVs via ultracentrifugation.

Invasion of HeLa Cells by Shigella Species Clinical Isolates Recovered from Pediatric Diarrhea.

Shigella is considered a major public health concern, especially for children younger than 5 years of age in developing countries. The pathogenicity of Shigella is a complex process that involves the interplay of multiple genes located on a large, unstable virulence plasmid as well as chromosomal pathogenicity islands. Since various factors (including virulence and antibiotic resistance genes) are associated with the severity and duration of shigellosis, in this article, we aim to evaluate whether the invasion of HeLa cells is affected by Shigella spp. isolates with different characteristics (including serogroups, virulence gene profiles, and antibiotic resistance patterns) recovered from pediatric patients in Tehran, Iran. Cell invasion ability of 10 Shigella isolates with different serogroups (Shigella flexneri and Shigella sonnei), gene profiling (virA, sen, ipgD, ipaD, ipaC, ipaB, and ipaH), and antibiotic resistance phenotyping (ampicillin, azithromycin, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, cefixime, cefotaxime, minocycline, and levofloxacin) were measured by plaque-forming assay in HeLa cell lines. The results show that all the selected Shigella spp. isolates recovered from pediatric patients were able to invade HeLa cells, but the total number and average size of plaques were different between the isolates. The higher invasion ability of S. flexneri isolates in HeLa cells compared to S. sonnei isolates was attributed to the presence of particular virulence genes; however, the role of each of these virulence factors remains to be determined.

Recombinant Cedar Virus: A Henipavirus Reverse Genetics Platform.

The isolation of Cedar virus, a nonpathogenic henipavirus that is closely related to the highly pathogenic Nipah virus and Hendra virus, provides a new platform for henipavirus experimentation and a tool to investigate biological differences among these viruses under less stringent biological containment. Here, we detail a reverse genetics system used to rescue two replication-competent, recombinant Cedar virus variants: a recombinant wild-type Cedar virus and a recombinant Cedar virus that express a green fluorescent protein from an open reading frame inserted between the phosphoprotein and matrix genes. This recombinant Cedar virus platform may be utilized to characterize the determinants of pathogenesis across the henipaviruses, investigate their receptor tropisms, and identify novel pan-henipavirus antivirals safely under biosafety level-2 conditions.

Simultaneous Short- and Long-Patch Base Excision Repair (BER) Assay in Live Mammalian Cells.

The base excision repair (BER) pathway repairs small, non-bulky DNA lesions, including oxidized, alkylated, and deaminated bases, and is responsible for the removal of at least 20,000 DNA lesions per cell per day. BER is initiated by DNA damage-specific DNA glycosylases that excise the damaged base and generates an abasic (AP) site or single-strand breaks, which are subsequently repaired in mammalian cells either by single-nucleotide (SN) or multiple-nucleotide incorporation via the SN-BER or long-patch BER (LP-BER) pathway, respectively. This chapter describes a plaque-based host cell reactivation (PL-HCR) assay system for measuring BER mechanisms in live mammalian cells using a plasmid-based assay. After transfection of a phagemid (M13mp18) containing a single modified base (representative BER DNA substrates) within a restriction site into human cells, restriction digestions detect the presence or absence (complete repair) of the adduct by the transformation of the digestion products into E. coli and counting the transformants as plaques. To monitor the patch size, different plasmids are constructed containing C:A mismatches within different restriction sites (inhibiting digestion) at various distances on both sides (5' or 3') of the modified base-containing restriction sites. Using this assay, the percentage of repair events that occur via 5' and 3' patch formation can be quantified.

Comparative characterization of Crimean-Congo hemorrhagic fever virus cell culture systems with application to propagation and titration methods.

Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is a biosafety level 4 and World Health Organization top priority pathogen. Infection leads to an often fatal hemorrhagic fever disease in humans. The tick-borne virus is endemic in countries across Asia, Europe and Africa, with signs of spreading into new regions. Despite the severity of disease and the potential of CCHFV geographic expansion to cause widespread outbreaks, no approved vaccine or treatment is currently available. Critical for basic research and the development of diagnostics or medical countermeasures, CCHFV viral stocks are commonly produced in Vero E6 and SW-13 cell lines. While a variety of in-house methods are being used across different laboratories, there has been no clear, specific consensus on a standard, optimal system for CCHFV growth and titration. In this study, we perform a systematic, side-by-side characterization of Vero E6 and SW-13 cell lines concerning the replication kinetics of CCHFV under different culture conditions. SW-13 cells are typically cultured in a CO2-free condition (SW-13 CO2-) according to the American Type Culture Collection. However, we identify a CO2-compatible culture condition (SW-13 CO2+) that demonstrates the highest viral load (RNA concentration) and titer (infectious virus concentration) in the culture supernatants, in comparison to SW-13 CO2- and Vero E6 cultures. This optimal viral propagation system also leads to the development of two titration methods: an immunostaining-based plaque assay using a commercial CCHFV antibody and a colorimetric readout, and an antibody staining-free, cytopathic effect-based median tissue culture infectious dose assay using a simple excel calculator. These are anticipated to serve as a basis for a reproducible, standardized and user-friendly platform for CCHFV propagation and titration.

Anti-HSV nucleoside and non-nucleoside analogues: spectroscopic characterisation of naphthyl and coumarinyl amides and their mode and mechanism of antiviral action.

Nucleoside analogues acyclovir, valaciclovir, and famciclovir are the preferred drugs against human Herpes Simplex Viruses (HSVs). However, the viruses rapidly develop resistance against these analogues which demand safer, more efficient, and nontoxic antiviral agents. We have synthesized two non-nucleoside amide analogues, 2-Oxo-2H-chromene-3-carboxylic acid [2-(pyridin-2-yl methoxy)-phenyl]-amide (HL1) and 2-hydroxy-1-naphthaldehyde-(4-pyridine carboxylic) hydrazone (HL2). The compounds were characterized by different physiochemical methods including elementary analysis, FT-IR, Mass spectra, 1H-NMR; and evaluated for their antiviral efficacy against HSV-1F by Plaque reduction assay. The 50% cytotoxicity (CC50), determined by MTT test, revealed that HL1 (270.4 µg/ml) and HL2 (362.6 µg/ml) are safer, while their antiviral activity (EC50) against HSV-1F was 37.20 µg/ml and 63.4 µg/ml against HL1 and HL2 respectively, compared to the standard antiviral drug Acyclovir (CC50 128.8 ± 3.4; EC50 2.8 ± 0.1). The Selectivity Index (SI) of these two compounds are also promising (4.3 for HL1 and 9.7 for HL2), compared to Acyclovir (49.3). Further study showed that these amide derivatives block the early stage of the HSV-1F life cycle. Additionally, both these amides make the virus inactive, and reduce the number of plaques, when infected Vero cells were exposed to HL1 and HL2 for a short period of time. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03658-0.

Mammalian Reoviruses: Propagation, Quantification, and Storage.

Mammalian reoviruses are pathogens that cause gastrointestinal and respiratory infections. In humans, the mammalian reoviruses usually cause mild or subclinical disease, and they are ubiquitous, with most people mounting immunity at a young age. Reoviruses are prototypic representations of the Reoviridae family, which contains many highly pathogenic viruses. This article describes techniques for culturing mouse fibroblast L929 cell lines, the preferred cell line in which most mammalian reovirus studies take place. In addition, mammalian reovirus propagation, quantification, purification, and storage are described. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Propagation of mammalian reoviruses in cell culture from virus stocks Alternate Protocol 1: Large-scale propagation (and purification) of mammalian reoviruses in cell culture from virus stocks Basic Protocol 2: Quantification of mammalian reoviruses by plaque assay with neutral red staining Alternate Protocol 2: Quantification of mammalian reoviruses by plaque assay with crystal violet staining Basic Protocol 3: Storage of mammalian reoviruses Support Protocol 1: Growth and maintenance of mouse L929 cells Support Protocol 2: Plating L929 cells.