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Food-borne virus:detection in a model system.

The purpose of this study was to develop methods for detection and quantitation of food-borne virus. Samples (25 g) of cottage cheese, contaminated with various quantities of coxsackievirus type A9, comprised the model system. Two of the methods presented have at least a 50% probability of detecting virus at levels below 5 plaque-forming units/25-g sample. Noteworthy aspects of these methods include use of a glycine-NaOH buffer (pH 8.8) containing approximately 1 m MgCl(2) as the diluent in which the sample is slurried, treatment of the slurry with Freon TF and bentonite to facilitate centrifuge clarification, and concentration of the clarified sample extract by a two-stage process employing polyethylene glycol followed by ultracentrifugation. Virus in the final 0.5-ml sample concentrate was detected and quantitated by the plaque technique in rhesus monkey kidney cell cultures. Processing of the sample requires approximately 2 days, and the inoculated cultures may have to be observed for as long as 7 days thereafter. If these levels of sensitivity are desired, and if 12 samples per day are tested on a routine basis, the cost savings achieved by employing these methods rather than testing sample extracts without concentration may range from 75 to 90%.

Buffers↗

Influenza virus detection in clinical specimens.

The authors compared the results of influenza A (H1N1) and influenza A (H3N2) virus detection in nasopharyngeal swabs from flu patients by molecular hybridization (MH), ELISA, virus isolation and seroconversion. Using the immunofluorescence (IF) technique influenza virus was detected in cell suspensions from the first chick embryo passage. Altogether 63 swabs from various epidemic seasons were separated into 3 groups according to specimen sampling and storage. It was shown that influenza virus RNA could be found in 16 out of 22 swab specimens (72%) stored at -70 degrees C without thawing and that ELISA revealed the influenza virus antigen in 19 cases (86%); in contrast, IF was positive in 6 (27%) and virus isolation in 5 (22%) cases only. However, the positive rate of MH decreased to 9% in 21 swab specimens repeatedly thawed and stored at -20 degrees C and was completely negative after prolonged storage of repeatedly thawed samples. Despite these conditions, ELISA was still successful in both latter sample groups (71-80%). For specificity control, 29 samples coming from patients with influenza B virus and other respiratory virus diseases (adeno- and respiratory syncytial virus) were used.

Antibodies, Viral↗

Whole Genome Sequencing and Genetic Diversity of Respiratory Viruses Detected in Children With Acute Respiratory Infections: A One-Year Cross-Sectional Study in Senegal.

Acute respiratory infections (ARI) are a health priority, especially in countries with limited resources. They are a major cause of morbidity and mortality, especially among children and the elderly. In Senegal, the endemic circulation of respiratory viruses other than influenza has been demonstrated. However, there is a paucity of data exploring the genetic diversity of these viruses based on whole-genome sequencing. In this study, we present data on the genetic diversity of respiratory viruses in children under 15 years old in Senegal, including an overview of the different pathogens detected. Between November 2022 and November 2023, we collected nasopharyngeal swabs from children seen in curative consultations for symptoms of acute respiratory infections. Of the 156 children included, 73.7% tested positive for at least one pathogen. The most frequently detected virus was rhinovirus (50.0%), followed by influenza B (41.6%) and human parainfluenza virus type 3 (7.6%). Combinations of rhinovirus/influenza B, human parainfluenza virus type 2/human parainfluenza virus type 4, and rhinovirus/influenza B/adenovirus were the most frequently identified. A statistically significant association was detected between some of the viruses detected. A high genetic diversity of respiratory viruses circulating in children was revealed. The strains were phylogenetically close to various strains circulating worldwide, suggesting a global circulation of respiratory viruses. Our study provides the first complete genome sequences of human parainfluenza viruses type 2, 3, 4 and human bocavirus from Senegal and thus contributes to the enrichment of international databases on sequences from Senegal and underlines the importance of sequencing in the dynamics of pathogen circulation.

Humans↗

Comparison of total culturable virus assay and multiplex integrated cell culture-PCR for reliability of waterborne virus detection.

The total culturable virus assay (TCVA) and an integrated cell culture-PCR (ICC-PCR) were compared in parallel to evaluate their detection reliability. Source, finished, and tap water samples from three drinking water treatment plant systems were analyzed by TCVA, and every cell culture dish was subsequently examined by reverse transcription (RT) multiplex PCR using enterovirus- and adenovirus-specific primers. Twenty-seven of 180 (15%) inoculated dishes exhibited cytopathic effects (CPE). Virus concentrations for source water ranged from 3.3 to 21.0 most probable numbers of infectious units (MPN) per 100 liters. No finished or tap water samples were positive. On the other hand, 38 (21%) of the dishes were positive in multiplex ICC-PCR. Virus concentrations ranged from 4.5 to 10.2 MPN/100 liters for source water and 0 to 0.9 MPN/100 liters for finished and tap water. In spite of its superior sensitivity, the ICC-PCR assay resulted in lower virus concentration values than the TCVA for two of the source water sites. Retest of the CPE-positive dishes using reovirus-specific RT-PCR revealed that 24 of the 27 (89%) dishes were also positive for reoviruses. These observations suggested that the detection reliability of ICC-PCR is restricted by the primer sets that are integrated in the reaction mixture. The observation of an uneven distribution of PCR-positive culture dishes in a given sample raises an additional caution that simple extrapolation of the ICC-PCR result from the analysis of a limited fraction of collected samples should be avoided to minimize possible over- and underestimation of the amount of virus.

Adenoviridae↗

Variation of the hepatitis C virus 5' non-coding region: implications for secondary structure, virus detection and typing. The International HCV Collaborative Study Group.

Variation in the 5' non-coding region (5'NCR) of hepatitis C virus (HCV) was investigated in detail by comparing 314 5'NCR sequences of viruses of genotypes 1 to 6. Evidence was obtained for the existence of associations between particular 5'NCR sequence motifs and virus types and subtypes. No recombination was observed between the 5'NCR and coding regions of different genotypes, implying that the sequence of subgenomic regions such as the 5'NCR can be used to deduce virus genotype. The distribution of polymorphic sites within the 5'NCR is used to propose improved oligonucleotide primers for virus detection and quantification that would be equally efficient in detecting RNA of different virus genotypes. The accuracy of two different genotyping methods (RFLP and the line probe assay) based on analysis of sequence polymorphisms in the 5'NCR is predicted from the sequences surveyed to be 97% and 83% respectively for types 1 to 6, with higher accuracies for distinguishing between subtypes 1a/1b, 2a/2b or 3a/3b. Several sites of genotype-specific polymorphism were covariant and maintained the base pairings required for a secondary structure model of the 5'NCR. Other sites of variation suggest minor modifications to this model and have implications for the probable functions of the 5'NCR.

Base Composition↗

Evaluation of linked PCR-transcription amplification procedure for bean yellow mosaic virus detection in gladioli.

Bean yellow mosaic virus (BYMV) concentration in in-vitro cultured Gladiolus cormlets was low and impossible to detect by the commonly used diagnostic methods. The polymerase chain reaction (PCR) detected viral RNA in most infected cormlets but not in all. Additional amplification of the PCR products by transcription, using T7 RNA polymerase (PCR/T), resulted in virus detection in cases which otherwise went undetected. PCR products having a single polymerase promoter at one end served as a better template for T7 RNA polymerase, and yielded more transcripts of a particular orientation than a template containing promoters at both ends. Repeated cycles of PCR/T resulted in the production of a heterogeneous amplified material which correlated with a progressive decline in amplification rate. Therefore, only the first PCR/T cycle proved to be effective. The PCR/T procedure was shown to be better than other commonly used diagnostic methods including PCR.

Enzyme-Linked Immunosorbent Assay↗

[Production of monoclonal antibodies to tomato mosaic virus and application in virus detection].

Four hybridoma cell lines secreting monoclonal antibodies(MAbs) against Tomato mosaic virus(ToMV) were produced by fusing mouse myeloma cells(SP2/0) with spleen cells from BAL B/c immunized by the ToMV particle. The four MAbs could specifically react with ToMV, and the MAbs from two cell lines can react with ToMV and TMV simultaneously. The titres of ascitic fluids of four MAbs ranged from 1:32,000 to 1:1,024,000 with ELISA, and the sensitivity for detection virus from the plant sap reached over 1:2000 dilution. The MAbs didn't crossreact with other plant viruses. The result of Western-blot showed that two MAbs can react with the 17.6 kD ToMV coat protein submit specifically, while the other two MAbs can not react with it, they are supposed to against conformational determinants of ToMV CP.

Animals↗

Enhanced parainfluenza I (6/94) virus detection in latently infected human brain cell cultures by treatment with cytochalasin D and dimethyl sulfoxide.

The ability of cytochalasin D (CD) and dimethyl sulfoxide (DMSO) to enhance parainfluenza I (6/94) virus replication was studied in various cell culture systems. Treatment of CV1 cells with CD (1 microgram/ml) dissolved in DMSO prior to primary 6/94 virus exposure at 10(0)--10(5) multiplicities of infection did not substantially enhance virus replication. However, there was a transient increase in cell associated virus one day after infection of DMSO-treated cultures. CD treatment of cultures of human brain cells latently infected with 6/94 virus (LIHB cells) did not enhance 6/94 virus detection. Cocultivation of CV1 cells with CD-treated LIHB cell cultures, and cocultivation of LIHB cell cultures with CD-treated CV1 cells, resulted in the production of both cell-associated and cell-free 6/94 virus three and five days after cocultivation. No virus was detected after similar cocultivation of untreated LIHB cell cultures with untreated CV1 cells. The usefulness of CD-DMSO treatment in the rescue of virus from 6/94 LIHB cell cultures appears limited to a cocultivation system. The use of these techniques to enhance virus rescue from human tissues suspected of harboring latent viral genomes is discussed.

Animals↗

[Participation of vaccinia virus in the pathogenesis of different clinical forms of postvaccinal complications. I. Frequency of vaccinia virus detection in the vaccinted who have usual and complicated reactions to vaccination].

The virological examination of 1365 samples taken from 469 children vaccinated against smallpox revealed considerable differences in the frequency and the time of vaccinia virus detection in different clinical forms of postvaccinal pathology as compared with uncomplicated vaccinal process. During the postvaccinal period taking its normal course vaccinia virus was isolated from 7.3% of children only from the pharynx till day 8 following vaccination. In generalized and creeping vaccinia the virus was isolated from 71.4% of children, in postvaccinal encephalitis from 57.1% of children, in vaccinal angina frove-mentioned complications vaccinia virus was detected in the samples obtained from the patients till days 24, 35, 15 and 24 respectively. The etiopathogenetic role of vaccinia virus in a number of postvaccinal complications is discussed.

Child, Preschool↗

SCAN: A sample-to-answer cross-priming isothermal assay for on-site virus detection with RT-qPCR sensitivity and genomically similar virus differentiation specificity.

Genomically similar viruses often differ in pathogenicity and host tropism due to specific mutations, and failure to distinguish them risks misdiagnosis and ineffective control. Molecular methods can differentiate such viruses but require laboratory settings and skilled personnel, while field-deployable immunological methods suffer from cross-reactivity. To address this challenge, we developed SCAN (Sample-to-answer Cross-priming isothermal amplification Assay with Nucleic acid strip), a general framework for on-site detection of genomically similar viruses. Comparative bioinformatics of isolation and sequencing data identifies key conserved differential determinants for primer design, ensuring specificity and reducing non-specific amplification. A one-tube cross-priming isothermal amplification (CPA) enables rapid target amplification without thermal cycling, and the products are visually detected on a nucleic acid strip. All steps are integrated into a handheld, lightweight device (9.9&#x202f;&#xd7;&#x202f;4.4&#x202f;&#xd7;&#x202f;3.3&#x202f;cm, <200&#x202f;g) that also prevents aerosol contamination. Using transmissible gastroenteritis virus (TGEV) and porcine respiratory coronavirus (PRCV), the latter a natural mutant of TGEV, as a model, SCAN achieves a detection limit of 102 copies/&#x3bc;L with sensitivity comparable to RT-qPCR and supports sample-to-answer testing within 80&#x202f;min and simple operations. With verified high sensitivity, specificity, and accuracy, as well as field usability, SCAN provides a generalizable route for developing point-of-care tests (PoCT) that require precise field differentiation of closely related pathogens.

Cross-priming isothermal amplification↗

Human immunodeficiency virus detection: correlation with clinical progression in the Edinburgh haemophiliac cohort.

HIV p24 antigenaemia and virus detection in cultures of peripheral blood lymphocytes were examined in 16 of 18 haemophiliacs infected with HIV by a single batch of Scottish National Blood Transfusion Service factor VIII concentrate. Six (38%) had p24 antigenaemia and 11 (69%) had positive lymphocyte cultures. All seven patients with serious HIV disease (CDC group IV) had positive lymphocyte cultures whereas four (57%) had p24 antigenaemia. Four of nine (44%) patients with asymptomatic HIV disease (CDC groups II and III) had positive cultures and two (22%) had p24 antigenaemia. Twenty-eight of 36 samples from the symptomatic group were HIV culture positive compared with nine of 30 samples from the asymptomatic group (P less than 0.001). None of 14 antibody negative haemophiliacs who also received the implicated batch of factor VIII had p24 antigenaemia or positive HIV cultures. The ability to detect HIV in cultured lymphocytes correlates with the clinical severity of HIV disease in this cohort.

AIDS Serodiagnosis↗

Adaptive virus detection using filament-coupled antibodies.

We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10(10) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.

Antigen-Antibody Complex↗

Influence of storage temperature on infectious hematopoietic necrosis virus detection by cell culture isolation and RT-PCR methods.

The detection of infectious hematopoietic necrosis virus (IHNV) in infected rainbow trout Oncorhynchus mykiss and in cell culture supernatants stored under different conditions was studied. IHNV-positive fish visceral organ homogenates and cell culture supernatants were incubated at 4 and 25 degrees C. Virus titre was measured by virus isolation on epithelioma papulosum cyprini (EPC) cells and the IHNV RNA was detected by RT-PCR and semi-nested RT-PCR. The influence of repeated freezing and thawing on the virus isolation from organ homogenates and from cell culture supernatants was studied as well. It was possible to isolate the virus from IHNV-positive organ material during the 3 d of incubation at 4 degrees C but, only on the first day of incubation at 25 degrees C. Viral RNA could be amplified during the incubation period of 35 d at 4 degrees C but only during 8 d of incubation at 25 degrees C. In IHNV-infected cell culture supernatant stored at 4 degrees C, it was possible to detect virus for 36 and 16 d in supernatant stored at 25 degrees C. Viral RNA could be followed by using molecular methods during the entire experimental period of 123 d. Each cycle of freezing and thawing of samples resulted in a reduction of IHNV titre in the suspension of visceral organs, while the virus titre in cell culture supernatant remained almost the same following 33 freezing-thawing cycles. The present results show that rapid laboratory processing and storage of potentially virus-containing tissue samples as well as the use of different detection methods are very important for efficient IHNV diagnosis.

Animals↗

Biotinylated nucleic acid hybridization probes for potato virus detection.

cDNA libraries, representative of potato viruses X (PVXc strain) and Y (PVY degrees strain) genomes were obtained. A PVX cDNA cloned fragment was sequenced and biotinylated to be used as hybridization probe for the detection of purified virus or nucleic acid extracts of infected plants. Dot hybridization assay was sensitive to detect 4 ng of viral particles, corresponding to about 200 pg of viral RNA. The level of detection in infected plant extracts was as effective as that obtained with the ELISA. The presence of biotinylated PVY cDNA in the hybridization mixture did not affect sensitivity of the PVX detection assay, suggesting that a single diagnostic assay for several potato viruses and virus-related pathogens could be developed.

Amino Acid Sequence↗

An improved dot-blot method for virus detection in chicken embryo fibroblast cultures.

A simplified dot-blot procedure is described for the detection of fowlpox virus (FPV) in infected monolayers of chicken embryo fibroblasts (CEF) cultured in 96-well microtiter plates. The relative resistance of DNA to hot NaOH, which hydrolyzes other macromolecules including RNA and protein, was exploited to solubilize virus infected cells and denature intracellular DNA in a simple, quick manner. Moreover, there was no need to purify virus or isolate viral DNA from cellular DNA prior to dot blotting. After incubation of CEF with FPV, the extracellular fluid from infected cells was collected for storage in 96-well microtiter plates. The remaining cell monolayers in each well were then solubilized with hot NaOH. The solubilized and denatured DNA was transferred to a nylon membrane using a dot-blot vacuum filtration manifold. Hybridization was carried out with a 32P-labeled FPV DNA probe. With this methodology it was possible to detect specific viral DNA sequences following the infection of cell monolayers with as little as 1 infectious unit per well. The ability to detect specific viral DNA sequences in infected cells, without the need to isolate pure viral DNA, made it possible to analyze large numbers of samples in a single experiment. Moreover, sufficient fowlpox virus was present in the extracellular media from each well for further amplification and analysis of selected samples.

Animals↗

A conformational epitope on the dimer of the fusion protein of respiratory syncytial virus detected in natural infections.

A murine monoclonal antibody (MAb), 2D8, was used in immunofluorescence reactions to detect respiratory syncytial virus (RSV) antigen in clinical specimens. Nasopharyngeal epithelial cells from 63 of 66 children with RSV infections reacted with this MAb. The MAb was further characterized and was demonstrated to recognize a conformational epitope on the dimer of the fusion protein of RSV. No reaction was detected with the MAb, 2D8, on Western blots of antigen prepared from RSV-infected HEp-2 cells under reducing conditions. Under non-reducing conditions, 2D8 reacted with a 145-170 K protein; this reactivity was lost when the antigen preparation was heated to 100 degrees C. 2D8 reacted with purified F glycoprotein of RSV Long in an ELISA, neutralized infectivity of RSV by >50% at a dilution of 1:500, and was able to inhibit cell-to-cell fusion of RSV-infected cells. In a competitive ELISA, the epitope detected by 2D8 was localized to antigenic site A. The conformational epitope detected by 2D8 required protein dimerization and glycosylation for full reactivity. This report extends previous characterizations of the F protein in its native state in that the MAb defines a conformational epitope on the fusion protein dimer that is expressed in natural infections and elicits antibody that can neutralize virus infectivity and inhibit cell-to-cell fusion. In addition to its application as a diagnostic reagent, this MAb can be of use in testing preparations of RSV or purified F protein in which the purification or extraction processes could have destroyed conformational epitopes.

Journal Article↗

Method for detecting viruses in aerosols.

A simple method with poliovirus as the model was developed for recovering human enteric viruses from aerosols. Filterite filters (pore size, 0.45 micron; Filterite Corp., Timonium, Md.) moistened with glycine buffer (pH 3.5) were used for adsorbing the aerosolized virus. No virus passed the filter, even with air flow rates of 100 liters/min. Virus recovery from the filter was achieved by rapid elution with 800 ml of glycine buffer, pH 10. The virus in the primary eluate was reconcentrated by adjusting the pH to 3.5, adding AlCl3 to 0.0005 M, collecting the virus on a 0.25-micron-pore Filerite disk (diameter, 25 mm) and and eluting with 6 ml of buffer, pH 10. With this method, virus could be detected regularly in aerosols produced by flushing when 3 X 10(8) PFU of poliovirus were present in the toilet bowl. Poliovirus-containing fecal material from two of four infants who had recently received oral polio vaccine also yielded virus in the aerosols when feces containing 2.4 X 10(7) to 4.5 X 10(7) PFU of virus had been added to the toilet bowl. Persons infected with a variety of natural enteric viruses are known to excrete this amount of virus in their daily stools.

Aerosols↗