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Modified vaccinia Ankara (MVA) virus is a widely used vaccine platform, making accurate titration essential for vaccination studies. However, the current plaque forming unit (PFU) assay, the standard for MVA titration, is prone to observer bias and other limitations that affect accuracy and precision. To address these challenges, we developed a new flow cytometry-based quantification method using a highly specific monoclonal antibody (mAb) for the detection of MVA-infected cells, as a more accurate titration assay. Through previous work, we serendipitously identified three MVA-specific hybridoma antibody clones, which we characterized through ELISA, immunoblot, and flow cytometry, confirming their specificity for MVA. Sequencing confirmed that each antibody was monoclonal, and mass spectrometry results revealed that all mAbs target the MVA cell surface binding protein (CSBP, MVA105L). We next optimized the titration protocol using the most effective mAb, 33C7 by refining culture conditions and staining protocols to enhance sensitivity and minimize background. Our optimized method demonstrated superior sensitivity, reliability, and reduced processing time when compared with the traditional PFU assay, establishing it as a more accurate and efficient approach for MVA titration. [ABSTRACT FROM AUTHOR]

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[Display omitted] The implementation of artificial intelligence-based systems (AI) into highly regulated industries faces significant regulatory and logistical challenges. The lack of established practices and guidelines, risk aversion attitude, perception of AI technology as insufficiently tested and unreliable are among the most important factors preventing wider adoption of AI solutions into pharmaceutical industry. Here we demonstrate by an example development and validation of neural network-based computer vision systems for binary classification of results from virus titration assays often used in research and production of vaccines and antivirals. The systems were shown to effectively classify images of cells from the titration assays to negative (non-infected) or positive (virus infected) with an accuracy of over 99%. The methods have been validated according to GMP principles and related guidelines. Regulatory and logistical challenges of implementation of AI-based solutions into GMP-compliant systems are discussed. Although the implementation of AI-systems into GMP-regulated environment brings specific challenges related to introduction of novel technologies, future benefits for early adopters could outweigh the initial efforts. [ABSTRACT FROM AUTHOR]

Rodents are widely used for the development of COVID-19-like animal models, the virological outcome being determined through several laboratory methods reported in the literature. Our objective was to assess the agreement between methods performed on different sample types from 342 rodents experimentally infected with SARS-CoV-2 (289 golden Syrian hamsters and 53 K18-hACE2 mice). Our results showed moderate agreement between methods detecting active viral replication, and that increasing viral loads determined by either RT-qPCR or infectious viral titration corresponded to increasing immunohistochemical scores. The percentage of agreement between methods decreased over experimental time points, and we observed poor agreement between RT-qPCR results and viral titration from oropharyngeal swabs. In conclusion, RT-qPCR and viral titration on tissue homogenates are the most reliable techniques to determine the presence and replication of SARS-CoV-2 in the early and peak phases of infection, and immunohistochemistry is valuable to evaluate viral distribution patterns in the infected tissues. [ABSTRACT FROM AUTHOR]

Rodents are widely used for the development of COVID-19-like animal models, the virological outcome being determined through several laboratory methods reported in the literature. Our objective was to assess the agreement between methods performed on different sample types from 342 rodents experimentally infected with SARS-CoV-2 (289 golden Syrian hamsters and 53 K18-hACE2 mice). Our results showed moderate agreement between methods detecting active viral replication, and that increasing viral loads determined by either RT-qPCR or infectious viral titration corresponded to increasing immunohistochemical scores. The percentage of agreement between methods decreased over experimental time points, and we observed poor agreement between RT-qPCR results and viral titration from oropharyngeal swabs. In conclusion, RT-qPCR and viral titration on tissue homogenates are the most reliable techniques to determine the presence and replication of SARS-CoV-2 in the early and peak phases of infection, and immunohistochemistry is valuable to evaluate viral distribution patterns in the infected tissues.

Avian influenza A H9N2 viruses are circulating in domestic poultry worldwide. In this research a low-pathogenic AIV H9N2 was multiplied on MDCK cell line and SPF eggs and irradiated by a Nor-dian gamma cell instrument. Irradiated and non-irradiated AIV samples were titrated by TCID50 and EID50 methods, respectively. Haemagglutinin antigen was analysed by Haemagglutinin test. Infectivi¬ty of irradiated virus samples was determined by cell culture and egg inoculation methods. The virus titration decreased as the dose of gamma radiation increased. AIV proliferation on cell culture can be inactivated by gamma irradiation at a lower dose of gamma-ray (20 kGy) than the virus inactivation on embryonated eggs (30 kGy). The safety test showed complete inactivation of AIV on allantoic fluid with gamma-ray doses: 30 kGy and 20 kGy for virus on MDCK cells after four blind cultures.

African swine fever is a viral disease of the family Suidae. Methods to detect and quantify African swine fever virus (ASFV) include qPCR and virus infectivity assays. Individual laboratories often use in-house procedures for these assays, which can hamper the comparison of results. The objective of this study was to estimate the probability of ASFV detection using these assays, and to determine the inter-test correlations between results. This was achieved by testing a panel of 80 samples at three reference laboratories. Samples were analysed using nucleic acid extraction and qPCR, as well as virus infectivity assays. For qPCR, a very high probability (ranging from 0.96 to 1.0) of detecting ASFV DNA was observed for all tested systems. For virus infectivity assays in cells, the probability of detecting infectious ASFV varied from 0.68 to 0.90 and was highest using pulmonary alveolar macrophages, followed by MARC145 cells, peripheral blood monocytes, and finally wild boar lung cells. Intraclass correlation coefficient estimates of 0.97 (0.96–0.98) between qPCR methods, 0.80 (0.74–0.85) to 0.94 (0.92–0.96) between virus infectivity assays, and 0.77 (0.68–0.83) to 0.95 (0.93–0.96) between qPCR methods and virus infectivity assays were obtained. These findings show that qPCR gives the highest probability for the detection of ASFV. [ABSTRACT FROM AUTHOR]

Reverse genetics has been used to generate recombinant Newcastle disease virus with enhanced immunogenic properties for vaccine development. The system, which involves co-transfecting the viral antigenomic plasmid with three helper plasmids into a T7 RNA polymerase-expressing cell to produce viral progenies, poses a great challenge. We have modified the standard transfection method to improve the transfection efficiency of the plasmids, resulting in a higher titer of virus progeny production. Two transfection reagents (i.e., lipofectamine and polyethylenimine) were used to compare the transfection efficiency of the four plasmids. The virus progenies produced were quantitated with flow cytometry analysis of the infectious virus unit. The modified transfection method increased the titer of virus progenies compared with that of the standard transfection method.

Seasonal influenza is an acute respiratory infection causing around 500 000 global deaths annually. There is an unmet medical need to develop more effective antiviral drugs and vaccines against influenza infection. A rapid, accurate, high‐throughput titration assay for influenza virus particles or neutralizing antibodies would be extremely useful in these research fields. However, commonly used methods such as tissue culture infective dose and plaque‐forming units (PFU) for virus particle quantification, and the plaque reduction neutralization test (PRNT) for antibody determination are time‐consuming, laborious, and have limited accuracy. In this study, we developed an efficient assay based on the enzyme‐linked immunospot (ELISPOT) technique for the influenza virus and neutralizing antibody titration. Two broad‐spectrum antibodies recognizing the nucleoproteins of influenza A and B viruses were used in the assay to broadly and highly sensitively detect influenza virus‐infected cells at 16 hours postinfection. An optimized cell culture medium with no tosyl phenylalanyl chloromethyl ketone trypsin and high dose oseltamivir acid was used to improve quantitation accuracy. This ELISPOT assay displayed a good correlation (R2 = 0.9851) with the PFU assay when used to titrate 30 influenza virus isolates. The assay was also applied to measure influenza‐neutralizing antibodies in 40 human sera samples, showing a good correlation (R2 = 0.9965) with the PRNT assay. This ELISPOT titration assay is a rapid, accurate, high‐throughput assay for quantification of influenza virus and neutralizing antibodies, and provides a powerful tool for research into and development of drugs and vaccines against influenza. Highlights: A rapid, accurate, high‐throughput assay for quantification of influenza virus and neutralizing antibodies based on the enzyme‐linked immunospot (ELISPOT) technique was developed.This assay displayed a good correlation (R2 = 0.9851) with the PFU assay for influenza virus titration.This assay also showed a good correlation (R2 = 0.9965) with the PRNT assay for neutralization antibody titer determination.This assay provides a powerful tool for research into and development of drugs and vaccines against influenza. [ABSTRACT FROM AUTHOR]

African swine fever is a viral disease of the family Suidae. Methods to detect and quantify African swine fever virus (ASFV) include qPCR and virus infectivity assays. Individual laboratories often use in-house procedures for these assays, which can hamper the comparison of results. The objective of this study was to estimate the probability of ASFV detection using these assays, and to determine the inter-test correlations between results. This was achieved by testing a panel of 80 samples at three reference laboratories. Samples were analysed using nucleic acid extraction and qPCR, as well as virus infectivity assays. For qPCR, a very high probability (ranging from 0.96 to 1.0) of detecting ASFV DNA was observed for all tested systems. For virus infectivity assays in cells, the probability of detecting infectious ASFV varied from 0.68 to 0.90 and was highest using pulmonary alveolar macrophages, followed by MARC145 cells, peripheral blood monocytes, and finally wild boar lung cells. Intraclass correlation coefficient estimates of 0.97 (0.96–0.98) between qPCR methods, 0.80 (0.74–0.85) to 0.94 (0.92–0.96) between virus infectivity assays, and 0.77 (0.68–0.83) to 0.95 (0.93–0.96) between qPCR methods and virus infectivity assays were obtained. These findings show that qPCR gives the highest probability for the detection of ASFV.

African swine fever virus (ASFV) is a complex DNA virus causing severe hemorrhagic disease in domestic pigs and wild boar. The disease has spread worldwide, with important socio-economic consequences. Early virus detection and control measures are crucial as there are no effective vaccines nor antivirals on the market. While the diagnosis of ASFV is fast and based primarily on qPCR, the detection of infectious ASFV is a labor-intensive process requiring susceptible macrophages and subsequent antibody-based staining or hemadsorption. The latter cannot detect ASFV isolates devoid of functional CD2v (EP402R) expression. Here, we report the development of a plasmid-based reporter assay (RA) for the sensitive detection and titration of infectious ASFV. To this end, we constructed a plasmid for secreted NanoLuc luciferase (secNluc) expression driven by the ASFV DNA polymerase gene G1211R promoter. Infection of plasmid-transfected immortalized porcine kidney macrophages (IPKM) followed by measurement of secNluc from cell culture supernatants allowed reliable automated quantification of infectious ASFV. The RA-based titers matched the titers determined by conventional p72-staining or hemadsorption protocols. The novel assay is specific for ASFV as it does not detect classical swine fever virus nor porcine reproductive and respiratory syndrome virus. It is applicable to ASFV of different genotypes, virulence, and sources, including ASFV from sera and whole blood from infected pigs as well as non-hemadsorbing ASFV. • This study describes a novel reporter assay for the titration of infectious ASFV with an automated luciferase readout. • The reporter assay detects a wide range of ASFV genotypes from different types of samples. • The reporter assay facilitates the detection and titration of non-hemadsorbing ASFV. • The luciferase readout offers potential for high-throughput research applications. [ABSTRACT FROM AUTHOR]

The titration of viruses onto susceptible cell lines is an important virological technique used to quantify infectious viral titers. It forms an integral component of epizootic hemorrhagic disease virus (EHDV) research, including estimating infectivity, calculating multiplicity of infection, and confirming virus propagation in cell culture. However, the ability to quantify infectious EHDV is also critical for disease control, particularly in the event of an outbreak. Routine EHD diagnostics do not accurately quantify infectious virus, which would allow accurate prediction of the onward transmission risk, but instead are typically more qualitative in nature (e.g., virus isolation) or only quantify viral genome copies (e.g., real-time PCR) which often remain detectable long after infectious virus is cleared from the host.Infectious EHDV titers are typically quantified through the detection of visible cytopathic effect (CPE) in the monolayer of susceptible mammalian cell cultures. However, not all susceptible cell lines demonstrate visible CPE upon EHDV infection, including cell lines such as KC cells, which are derived from the EHDV biological insect vector, Culicoides sonorensis. This chapter presents a comprehensive method for the titration of EHDV-positive samples onto relevant, susceptible mammalian (Vero) and insect (KC) cell lines and describes alternative methods that can be used to visualize EHDV infection, by CPE or immunofluorescent labeling of viral proteins, to enable the calculation of infectious EHDV titers., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are members of the Herpesviridae family. About 40% to 80% of the world populations are infected with HSV and its prevalence is high in Iran. The high prevalence of this virus in the community and the ability of the virus in causing fatal diseases among immunocompromised patients, have encouraged studies to be performed on HSV and suitable cell lines which supports the propagation of HSV. The aim of this study was to evaluate the suitability of McCoy cell line in the isolation and propagation of HSV. An isolated wild-type HSV-1 was obtained from the labial vesicles of a 29-year-old patient who was referred to Ghaem Hospital (Mashhad, Iran). The virus was inoculated in McCoy cell monolayer cells and its titer was calculated by 50% tissue culture infectious dose (TCID50) method. Cytopathic effects (CPE) of HSV on McCoy cells appeared about 20 hours after the infection of cells. Titer of the virus was 10-5.25 TCID50/ml. Our data showed that the McCoy cell line supported the propagation of HSV in high titer. This was the first study that used McCoy cell line for the isolation and propagation of HSV-1. McCoy cell line could be used, as a proper cell line of HSV, for various studies in the future.

Among several strategies used for Herpes simplex virus (HSV) detection in biological specimens, standard plaque assay (SPA) remains the most reliable method to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may be particularly laborious for multiple sample analysis. Here we describe an innovative method to perform the titration of HSV type 1 (HSV-1) in different samples, using the “In-Cell WesternTM” Assay (ICW) from LI-COR, a quantitative immunofluorescence assay that exploits laser-based scanning of near infrared (NIR). In particular, we employed NIR-immunodetection of viral proteins to monitor foci of HSV-1 infection in cell monolayers, and exploited an automated detection of their fluorescence intensity to evaluate virus titre. This innovative method produced similar and superimposable values compared to SPA, but it is faster and can be performed in 96 well plate, thus allowing to easily and quickly analyze and quantify many samples in parallel. These features make our method particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1 drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving data reproducibility. Overall, our method may represents a useful tool for both clinical and research purposes.

The classical cell-culture methods, such as cell culture infectious dose 50% (CCID 50 ) assays, are time-consuming, end-point assays currently used during the development of a viral vaccine production process to measure viral infectious titers. However, they are not suitable for handling the large number of tests required for high-throughput and large-scale screening analyses. Impedance-based bio-sensing techniques used in real-time cell analysis (RTCA) to assess cell layer biological status in vitro , provide real-time data. In this proof-of-concept study, we assessed the correlation between the results from CCID 50 and RTCA assays and compared time and costs using monovalent and tetravalent chimeric yellow fever dengue (CYD) vaccine strains. For the RTCA assay, Vero cells were infected with the CYD sample and real-time impedance was recorded, using the dimensionless cell index (CI). The CI peaked just after infection and decreased as the viral cytopathic effect occurred in a dose-dependent manner. The time to the median CI (CIT med ) was correlated with viral titers determined by CCID 50 over a range of about 4–5 log 10 CCID 50 /ml. This in-house RTCA virus-titration assay was shown to be a robust method for determining real-time viral infectious titers, and could be an alternative to the classical CCID 50 assay during the development of viral vaccine production process. [ABSTRACT FROM AUTHOR]

A novel biopolymeric material in the form of nano/microspheres was developed which was capable of adsorbing coronaviruses. The biopolymer was obtained by crosslinking of chitosan (CHIT) with genipin, a nontoxic compound of plant origin, in inverted emulsion and reacting the chitosan nano/microspheres obtained (CHIT-NS/MS) with glycidyltrimethyl-ammonium chloride (GTMAC). As a result the nano/microspheres of N-(2-hydroxypropyl)-3-trimethyl chitosan (HTCC-NS/MS) were obtained. HTCC-NS/MS were studied as the adsorbents of human coronavirus NL63 (HCoV-NL63), mouse hepatitis virus (MHV), and human coronavirus HCoV-OC43 particles in aqueous virus suspensions. By studying cytopathic effect (CPE) caused by these viruses and performing PCR analyses it was found HTCC-NS/MS strongly adsorb the particles of HCoV-NL63 virus, moderately adsorb mouse hepatitis virus (MHV) particles, but do not adsorb HCoV-OC43 coronavirus. The adsorption capacity of HTCC-NS/MS well correlated with the antiviral activity of soluble HTCC against a given virus. Importantly, it was shown that HCoV-NL63 particles could be desorbed from the HTCC-NS/MS surface with a salt solution of high ionic strength with retention of virus virulence. The obtained material may be applied for the removal of coronaviruses, purification and concentration of virus samples obtained from biological matrices and for purification of water from pathogenic coronaviruses. [ABSTRACT FROM AUTHOR]

Among several strategies used for Herpes simplex virus (HSV) detection in biological specimens, standard plaque assay (SPA) remains the most reliable method to evaluate virus infectivity and quantify viral replication. However, it is a manual procedure, thereby affected by operator subjectivity, and it may be particularly laborious for multiple sample analysis. Here we describe an innovative method to perform the titration of HSV type 1 (HSV-1) in different samples, using the "In-Cell WesternTM" Assay (ICW) from LI-COR, a quantitative immunofluorescence assay that exploits laser-based scanning of near infrared (NIR). In particular, we employed NIR-immunodetection of viral proteins to monitor foci of HSV-1 infection in cell monolayers, and exploited an automated detection of their fluorescence intensity to evaluate virus titre. This innovative method produced similar and superimposable values compared to SPA, but it is faster and can be performed in 96 well plate, thus allowing to easily and quickly analyze and quantify many samples in parallel. These features make our method particularly suitable for the screening and characterization of antiviral compounds, as we demonstrated by testing acyclovir (ACV), the main anti-HSV-1 drug. Moreover, we developed a new data analysis system that allowed to overcome potential bias due to unspecific florescence signals, thus improving data reproducibility. Overall, our method may represents a useful tool for both clinical and research purposes. [ABSTRACT FROM AUTHOR]

Hepatitis A virus (HAV) replicates relatively slowly in cell culture without a cytopathic effect, a fact that limits the use of tissue culture assays. The radioimmunofocus assay is the standard method for HAV titration, although it is labor intensive and requires the use of radioisotopes. A simple, rapid and objective infectivity assay based on an in situ enzyme immunoassay (EIA) is described here for a Brazilian cell culture-adapted HAV strain (HAF-203). The assay uses a peroxidase-labeled polyclonal antibody to fixed monolayers as an indicator of infection. EIA may be completed within 7 days using serial 5-fold dilutions of the virus, yielding a titer of 5.024 log 50% tissue culture infective dose (TCID50)/ml for HAF-203. This technique had a detection limit of 1.1 log TCID50/ml and the specificity was demonstrated by detecting no reaction on the columns of uninfected wells. The reproducibility (with intra- and inter-assay coefficients of variation ranging from 1.9 to 3.8% and from 3.5 to 9.9%, respectively) and quantitation of the assay were demonstrated by close agreement in virus infectivity titers among different assays of the same amount of virus and between assays of different amounts of virus. Furthermore, this assay does not require the use of radiolabeled antibodies. We describe here an efficient EIA that is highly reproducible and that could be used to monitor HAV growth in cell culture and to determine the quantity of HAV antigen needed for diagnostic assays. This is the first report of the infectious titer of the Brazilian cell culture-adapted HAV strain (HAF-203).

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) are members of the Herpesviridae family. About 40% to 80% of the world populations are infected with HSV and its prevalence is high in Iran. The high prevalence of this virus in the community and the ability of the virus in causing fatal diseases among immunocompromised patients, have encouraged studies to be performed on HSV and suitable cell lines which supports the propagation of HSV. The aim of this study was to evaluate the suitability of McCoy cell line in the isolation and propagation of HSV. An isolated wild-type HSV-1 was obtained from the labial vesicles of a 29-year-old patient who was referred to Ghaem Hospital (Mashhad, Iran). The virus was inoculated in McCoy cell monolayer cells and its titer was calculated by 50% tissue culture infectious dose (TCID50) method. Cytopathic effects (CPE) of HSV on McCoy cells appeared about 20 hours after the infection of cells. Titer of the virus was 10-5.25 TCID50/ml. Our data showed that the McCoy cell line supported the propagation of HSV in high titer. This was the first study that used McCoy cell line for the isolation and propagation of HSV-1. McCoy cell line could be used, as a proper cell line of HSV, for various studies in the future. [ABSTRACT FROM AUTHOR]

Classical swine fever (CSF) is a highly contagious notifiable disease of pigs caused by CSF virus of Flaviviridae family. Previously, lapinized vaccines were used for the disease control, which has now been replaced with cell culture vaccines. Determination of virus titre is the key factor for development and quality control testing of classical swine fever (CSF) cell culture vaccines. Since CSFV is a non- cytopathic virus, an accurate method for the titration of this virus in cell culture has not yet been reported. Here we present a full proof method of titration of CSF cell culture viruses employing Fluorescent Antibody Technique (FAT) in 24 well plate cover slip culture of PK-15 cells. CSFV monoclonal antibodies (Mab) used in the test bind to the CSF virus particles in the cell cytoplasm of the infected cells and the immune-fluorescence signal is produced by subsequent binding of FITC conjugate with Mab. In this newly developed method, apple green fluorescence is observed in the cytoplasm of the infected cells as the virus multiplies only in the cytoplasm. The nucleus as well as the uninfected cells cytoplasm is stained red without any traces of green fluorescence. Thus, the test clearly differentiates a CSFV infected cell from the uninfected cells in the vicinity, if any, and also from the uninfected controls. The test can also quantify the accurate titres of CSF live viruses in the cell culture vaccines and hence it has wide application in routine virus titration applied for manufacturing of CSF cell culture vaccines, determination of accurate multiplicity of infection (m.o.i.) during infection and quality control of vaccines by the testing laboratories. Schematic representation of the steps followed for virus titration in cover-slip cultures in 24 well plate and performing FAT staining of the infected cells. [Display omitted] [ABSTRACT FROM AUTHOR]

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., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Newcastle disease (ND) is one of the highly pathogenic viral diseases of avian species. The disease is endemic in many developing countries where agriculture serves as the primary source of national income. Newcastle disease virus (NDV) belongs to the family Paramyxoviridae and is well characterized member among the avian paramyxovirus serotypes. The failure of vaccination is one of the major causes of NDV outbreaks in field condition. The present study gives a brief picture about the biology of NDV genome and its proteins under different conditions of temperature and pH. Our results indicate that the NDV is non-infective above 42 °C and unstable above 72 °C. The study will be useful in defining an optimum storage condition for NDV without causing any deterioration in its viability. [ABSTRACT FROM AUTHOR]

Highlights: [•] A qRT-PCR method for the titration of Semliki Forest virus was standardized. [•] The method allowed the determination of temperature degradation of SFV. [•] Good correlations between MOI and recombinant protein expression were found. [ABSTRACT FROM AUTHOR]

The 50 % tissue culture infectious dose (TCID) is still one of the most commonly used techniques for estimating virus titers. However, the traditional TCID assay is time consuming, susceptible to subjective errors and generates only quantal data. Here, we describe a colorimetric-based approach for the titration of Enterovirus 71 (EV71) using a modified method for making virus dilutions. In summary, the titration of EV71 using MTT or MTS staining with a modified virus dilution method decreased the time of the assay and eliminated the subjectivity of observational results, improving accuracy, reproducibility and reliability of virus titration, in comparison with the conventional TCID approach ( p < 0.01). In addition, the results provided evidence that there was better correlation between a plaquing assay and our approach when compared to the traditional TCID approach. This increased accuracy also improved the ability to predict the number of virus plaque forming units present in a solution. These improvements could be of use for any virological experimentation, where a quick accurate titration of a virus capable of causing cell destruction is required or a sensible estimation of the number of viral plaques based on TCID of a virus is desired. [ABSTRACT FROM AUTHOR]

Abstract: The baculovirus-insect cell expression system is an important technology for the production of recombinant proteins and baculovirus-based biopesticides. Budded virus titration is critical when scaling up baculovirus production processes in suspension cultures, to ensure reproducible infections, especially when a low multiplicity of infection (MOI) is applied. In this study, a simple suspension culture titration (SCT) assay was developed that involves accurate measurements of the initial cell densities (ICDs) and peak cell densities (PCDs) of an infected culture, from which the MOI and hence the virus inoculum infectious titer can be estimated, using the established Power–Nielsen baculovirus infection model. The SCT assay was assessed in parallel with two adherent culture-based assays (MTT and AlamarBlue) for the Heliothine baculovirus HaSNPV, and was shown to be more objective, time-efficient and reproducible. The model predicted a linear correlation between log(PCD/ICD) and log(MOI), hence an alternative model-independent SCT assay was also developed, which relies on a well-replicated standard curve relating suspension culture-derived PCD/ICD ratios with plaque or endpoint assay-derived MOIs. Standard curves with excellent linearity were generated for HaSNPV and the industrially significant rAcMNPV, demonstrating the feasibility of this simple titration approach, especially in terms of its applicability to a wide range of virus infection kinetics. [Copyright &y& Elsevier]

Abstract: Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence™, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC50) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced. [ABSTRACT FROM AUTHOR]

Abstract: Different naturally occurring, cell adapted or genetically manipulated stocks of African swine fever virus were able to infect directly cultures of COS-1 cells, producing extensive cytopathic effects and amounts from 106 to 107 of infective progeny virus per ml. The induction of late virus-specific proteins, demonstrated by RT-PCR and immunoblotting, and the development of lysis plaques by all the virus samples tested so far, allowed the optimization of both titration and diagnostic assays, as well as the proposal of a method for selection of virus clones during the generation of virus mutants with specific gene deletions. [Copyright &y& Elsevier]

Abstract: Peste des petits ruminants (PPR) is a highly contagious and economically important viral disease of goats and sheep. A homologous Vero cell-based attenuated PPR vaccine developed in our laboratory and used extensively throughout the country, is available for control of PPR. The presently used quality control test, titration in Vero cells for PPR virus titre in vaccine batches, takes at least 6–8days to determine the quality and dose of vaccine. In this study, 74 freeze-dried PPR vaccine batches were tested simultaneously by both virus titration and PPR sandwich ELISA (S-ELISA) to correlate the titre of the vaccine virus with reactivity in S-ELISA. It was found that the vaccine batches with titre more than 103 TCID50/ml gave positive results in S-ELISA and correlated well with the virus titre of the freeze-dried vaccines. The correlation coefficient between the virus titration and S-ELISA reactivity was estimated as 0.96, indicating a high correlation between the two parameters based on 74 batches of freeze-dried PPR vaccine. The vaccine batches with titres of 3.0, 4.3, 4.5, 5.0, 6.5 and 7.0 had shown a positive reaction when tested in two-fold dilutions in S-ELISA at 1, 5, 6, 7, 8 and 9log2 titres, respectively. The test vaccine batches were found to be negative in S-ELISA when the titre of the vaccine was less than 103 TCID50/ml, suggesting that the vaccine could not be passed for field use. It is concluded that S-ELISA could be a preliminary tool useful for the quality control of PPR vaccine as it is rapid and easy to perform when compared to virus titration. [Copyright &y& Elsevier]

Abstract: India is endemic for foot-and-mouth disease and it continues to be a major threat to the livestock industry despite vaccination programmes. In the present study, the ability of specific small interfering (si)RNAs directed against different genomic regions of foot-and-mouth disease virus (FMDV) to inhibit virus replication in BHK-21 cells was examined. For preliminary evaluation of possible siRNA-mediated FMDV inhibition, a cocktail of several unique populations of 12–30bp siRNAs were successfully produced corresponding to three target regions located at structural (VP3-VP1), non-structural (2A-2C), and non-structural-untranslated (3D-3′UTR) region of serotype Asia1. Once the populations of siRNAs generated were found to reduce the virus titre significantly, two highly conserved 21bp siRNA duplexes were designed by analysing all FMDV sequence entries available in public-domain databases. In virus titration assay, more than 99% inhibition of virus yield for all the four serotypes (type Asia1, O, A, and C) could be demonstrated in cells transfected with each of the FMDV-specific siRNAs at 24h post-infection, compared to control cells transfected with scrambled siRNA. This was well supported by reduction in OD values in FMDV-specific sandwich ELISA. Although 100-fold reduction in virus titre with siRNA1 is substantial considering the transfection efficiency and fixed level of input siRNA, siRNA2 emerged to be a better choice as target where more than 300-fold reduction was observed and its inhibitory effect extended up to 48h post-infection against all the serotypes. Interestingly, in the present study type A virus (IND 17/77) had a single mismatch at position 2 in the siRNA2 target region but it did not abrogate the inhibitory effect. [Copyright &y& Elsevier]

Avian influenza (AI) viruses are endemic in wild birds and if transmitted to poultry can cause serious economic losses. In the study of AI, the quantitation of virus shed from infected birds is valuable in pathogenesis studies and to determine the effectiveness of vaccines, and is performed routinely by cultivation of virus containing samples using embryonating chicken eggs (ECE) and expressed by 50% egg infectious dose (EID50). Although, this assay is accurate and is the standard test for infectious virus titration, the method is laborious, requires a large number of ECE, and takes at least 7 days to determine results. In this study, a one-tube hydrolysis fluorescent probe based real-time RT-PCR (RRT-PCR) was applied for the quantitation of AI virus and compared with conventional virus titration method. A strong positive correlation was observed between the amount of RNA determined by quantitative RRT-PCR and the EID50s determined by conventional methods. This RRT-PCR test was further applied in the study of competitive replication of co-infected H5 and H7 subtype viruses in chickens. Using hemagglutinin subtype specific probes, we were able to determine the amount of individual subtype virus, which could not have easily been done with conventional methods. This RRT-PCR based quantitation of AI virus, which is specific, sensitive, easy to perform, and rapid, will be useful for virological, pathogenesis, and protection studies. [Copyright &y& Elsevier]

A 17-mer peptide (SAAEASAKATAEATAKG, p5) was selected by screening a combinatorial library for its ability to enhance in vitro the infectivity of viral hemorrhagic septicemia virus (VHSV), a salmonid rhabdovirus. Preincubation of VHSV samples with p5 at micromolar concentrations led to up to 5-fold increase of viral titers compared to untreated samples, as measured by a 1-day post-infection immunochemical focus assay. Treatment with p5 also increased VHSV titers when using the more traditional plaque and end-point dilution assays. Preincubation of p5 with infectious hematopoietic necrosis virus (another rhabdovirus of salmonids), but not with infectious pancreatic necrosis virus (birnavirus) also led to a similar increase in sensitivity. These results indicate that the addition of p5 may be used to improve the sensitivity of diagnostic tests for salmonid rhabdoviruses. [Copyright &y& Elsevier]

• HCoV-OC43 grows to a higher titer, sooner in human lung fibroblasts (MRC-5) than in human ileocecal adenocarcinoma cells (HRT-18). • Optimization of an HCoV-OC43 antibody-based TCID 50 assay reduced cost by 67 % and time by two hours. • At peak titer the defective virion to infectious virus ratio was <3:1 and nearly 100:1 for MRC-5 and HRT-18 cells, respectively. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-CoV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus , targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose 50 (TCID 50)/mL or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID 50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ˜107 plaque forming units/mL at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ˜106.5 TCID 50 /mL. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID 50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts. [ABSTRACT FROM AUTHOR]

• The here described method allows to distinguish MLV/MSCV vector RNA from transfer plasmid and proviral DNA. • The LOD 95% was determined and is 25 copies per qPCR reaction for DNA and 80 copies per RT-qPCR reaction for RNA. • The method has been shown to be robust and selective. • The primer and probe combination can be transferred to digital droplet PCR (ddPCR), enabling standard free quantification. Murine leukemia virus (MLV) and murine stem cell virus (MSCV) and derived retroviral vectors are widely used to study retrovirus biology and as tools for gene delivery. The method described here represents a quantitative real time PCR (qPCR) with hydrolysis probe that can be applied within classical qPCR as well as in digital droplet PCR (ddPCR). The method targets a 60 bp long fragment located within the U5 region of the MLV/MSCV genome sequence. For the here described method a LOD95% of 25 copies per PCR reaction (DNA) and 80 copies per PCR reaction (RNA) was determined, and PCR efficiencies of 92.5 % and 98.5 %, respectively, were observed. This method enables the fast and simple titration of viral genomic RNA present in retroviral vector stocks for accurate and consistent transduction experiments. Furthermore, it enables the detection of proviral and transfer plasmid derived DNA sequences and can be modified to differentiate between retroviral RNA and DNA. [ABSTRACT FROM AUTHOR]

The present study, developed in the scope of a survey to monitor enteric viruses in natural surface water and drinking water sources, addressed the suitability of mengovirus to assess viral recovery rates at two steps of the water sampling process. In a pilot campaign comprising two samples from each type of water source, when mengovirus was added after the filtration/primary concentration step, the recovery rates of viral RNA were higher than 18% and identical for both water matrices. In a one-year sampling campaign, where mengovirus was present along the whole sample processing (addition in the filtration/primary concentration step), significantly different recovery rates were observed between water matrices: usually higher than 1% in drinking water and under 1% in surface water. The results suggest the first stage of the water sampling process and the type of water matrix are the most influential factors for viral RNA recovery. This study also addressed and evidenced mengovirus replication and titration in Vero E6 cultures and showed infectious mengovirus to be recovered from samples of both types of water matrix. These results anticipate a more comprehensive applicability of mengovirus as a process control virus in the monitoring of viruses in water, extended to viral infectivity. [ABSTRACT FROM AUTHOR]

The ability to manipulate capripoxvirus through gene knockouts and gene insertions has become an increasingly valuable research tool in elucidating the function of individual genes of capripoxvirus, as well as in the development of capripoxvirus-based recombinant vaccines. The homologous recombination technique is commonly used to generate capripoxvirus knockout viruses (KO), and is based on the targeting of a particular viral gene of interest. This technique can also be used to insert a gene of interest. A protocol for the generation of a viral gene knockout is described. This technique involves the use of a plasmid which encodes the flanking sequences of the regions where the homologous recombination will occur, and will result in the insertion of an EGFP reporter gene for visualization of recombinant virus, as well as the E. coli gpt gene as a positive selection marker. If an additional gene is to be incorporated, this can be achieved by inserting a gene of interest for expression under a poxvirus promoter into the plasmid between the flanking regions for insertion. This chapter describes a protocol for generating such recombinant capripoxviruses. An alternative step for the removal of both the EGFP and gpt cassettes and an optional selection step using CRISPR technology are also described., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective To evaluate the effect of time, temperature and storage vial material on the antiviral activity of 0.5% cidofovir solution. Procedures Commercial 7.5% cidofovir solution for injection was diluted with normal saline to a 0.5% concentration. Aliquots were stored in plastic and glass vials at 4, −20, and −80 °C for 30, 60, 120, and 180 days. Antiviral activity against feline herpesvirus was evaluated in a virus titration assay at time zero (baseline) and at each subsequent time point. Results Cidofovir caused a fourfold log reduction in virus titer at baseline and at each time point and for each storage condition ( P < 0.001). Conclusion 0.5% cidofovir demonstrated stable antiviral activity when stored for up to 6 months in glass or plastic, at 4, −20, and −80 °C. [ABSTRACT FROM AUTHOR]

Chicken tracheal organ cultures (TOCs) provide a simple ex vivo system that makes use of transverse section of tracheal rings extracted from embryos or adult birds to perform classical virological techniques for virus isolation, propagation and titrations, alongside with gene-expression analysis and virus-host interaction studies. Most IBV strains replicate well in TOCs, thus conveniently allowing growth kinetics analysis. Viral replication is revealed by observation of ciliostasis as marker of infection in tracheas extracted from birds ex vivo, as well as in vitro analysis providing a reliable infection model and a useful tool for titration.