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A common method for cell-associated virus isolation involves disruption of infected cells by a combination of hypotonic burst, freeze-thaw cycles (F-T) and sonication. This protocol was also originally used for the preparation of cell-free extract containing the MX strain of lymphocytic choriomeningitis virus (LCMV), which is preferentially propagated by cell-to-cell contact and does not release distinct virions into the medium. In the present study, we compared different approaches to virus isolation. Based on virus yield, we show that deionized water lysis is the fastest and most effective method for releasing LCMV MX infectious viral particles from persistently infected cells. Moreover, we demonstrate that freeze-thaw cycles and sonication do not improve virus isolation. This simple protocol could be used for isolation of other viruses, the life cycle of which is strictly cell-associated and therefore are difficult to release in large amounts from host cells., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Label-free technology-based Quartz Crystal Microbalance (QCM) is an emerging tool in biological research. In this study, QCM was applied successfully for the rapid diagnosis of H3N2 canine influenza virus (CIV) infection. ProLinker™ B, a calixcrown derivative, enables antibodies to be attached to a gold-coated quartz surface and positioned in a regular pattern with the correct orientation. The ProLinker-coated quartz-based assay detected H3N2 CIV at lower concentrations (2(2) HA unit) than a commercial immunochromatography Ag kit (2(3) HA unit). Three independent experiments in which H3N2 CIV-positive reference samples were applied to an anti-CIV nucleoprotein (NP) monoclonal antibody immobilized on a quartz surface yielded standard deviations (SD) of ≤5%, indicating high reproducibility. In addition, the QCM assay with a cut-off value (-140 Hz) showed 97.1% (34/35) sensitivity and 94.7% (36/38) specificity in testing 73 field saliva samples, respectively. Thus, the QCM assay described herein will be a valuable tool for the rapid diagnosis of H3N2 CIV infection with high sensitivity and specificity, and should overcome several of the disadvantages and limitations inherent in the commercial immunochromatography Ag kit., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25×10(2) and 2.40×10(4) plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cost effective diagnostic tests are needed in rabies virus (RABV) enzootic areas to study the prevalence, distribution, and transmission of rabies virus among reservoir hosts. To reduce the associated costs of acquiring and maintaining specialized laboratory equipment, an indirect rapid immunohistochemistry test (IRIT), for the detection and differentiation of RABV variants, was evaluated by traditional light microscopy. The IRIT utilizes fresh frozen brain touch impressions or cell culture monolayers fixed in buffered formalin, a panel of murine anti-nucleoprotein monoclonal antibodies (mAb-N) and commercially available biotin-labeled goat anti-mouse antibody. In this study, 96 RABV isolates, representing 20 RABV variants previously determined by antigenic typing using a panel of mAb-N and the indirect fluorescent antibody test (IFA), and genetic sequence analysis were characterized by IRIT and the results compared. The IRIT results revealed distinct reactivity patterns associated with current and historical RABV reservoir hosts similar to IFA test and genetic sequence analysis. Evaluation of suspected RABV samples through IRIT does not require specialized equipment and is possible to perform in a field setting. Additionally, commercially available labeled secondary antibodies permit the use of a standard panel of unlabeled primary mAbs, without the need for fluorescence microscopy, and should augment existing attempts at antigenic characterization during canine rabies elimination campaigns in developed and developing countries. These results are useful in studying the epizootiology of rabies and inferring the source of infection when unknown., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Lymphocystis disease virus (LCDV) is the causative agent of lymphocystis disease affecting marine and freshwater fish worldwide. Here an antibody microarray was developed and employed to detect LCDV in fish. Rabbit anti-LCDV serum was arrayed on agarose gel-modified slides as capture antibody, and Cy3-conjugated anti-LCDV monoclonal antibody (MAbs) was added as detection antibody. The signals were imaged with a laser chip scanner and analyzed by corresponding software. To improve the sensitivity, different substrate binders (poly-L-lysine, MPTS, aldehyde, APES and agarose gel modified slides, and commercially available amino-modified slides), markers (fluorescein isothiocyanate, Cy3, horseradish peroxidase, biotin or colloidal gold) conjugated to anti-LCDV Mabs, and storage time of the antibody were assessed. The results showed that the antibody microarrays based on agarose gel-modified slides gave a lower detection limit of 0.55μg/ml of LCDV when Cy3 and HRP conjugated anti-LCDV MAbs were used as detection antibody; and the lowest detectable LCDV protein concentration was 0.0686 μg/ml when streptavidin-biotin conjugated to anti-LCDV MAbs served as detection antibody. The developed antibody microarray proved to have a high specificity for LCDV detection and a shelf-life of more than 8 months at -20°C. Furthermore, the LCDV detection results of the microarray in fish gills or fins (n=50) presented a concordance rate of 100% with enzyme-linked immunosorbent assay (ELISA) and 98% with immunofluorescence assay technique (IFAT). These results revealed that the developed antibody microarray could serve as an effective tool for diagnostic and epidemiological studies of LCDV in fish., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A rapid and simple immunochromatography strip (ICS) test for the specific detection of vesicular stomatitis virus serotype Indiana (VSV-IND) using two distinct monoclonal antibodies MAbs (1A2 and 4C3) against the G protein of VSV-IND was developed. The MAb 1A2 was conjugated with colloidal gold, and the MAb 4C3 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated T and C, respectively. The results showed that samples of VSV-IND combined with CG-MAb 1A2, and that these complexes were captured by MAb 4C3 at the test line (T), resulting in the appearance of a purple band. When samples did not contain VSV-IND or when they contained a quantity of VSV-IND below the detection limit of the test, only the control line (C) was visible. The analysis of the sensitivity of the test demonstrated that the lowest detected quantity of VSV-IND was 1.85×10(3.0)TCID50/ml. Storage of the ICS test at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. In clinical trials using RT-PCR as a reference test, the relative specificity and sensitivity of the ICS were determined to be 98.9% and 91.4%, respectively. Based on these results, the ICS test developed may be a suitable tool for rapid on-site testing for VSV-IND infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) employing monoclonal and polyclonal antibodies against p27 was developed for the detection of the avian leukosis virus (ALV). The specificity of the optimized AC-ELISA was evaluated using avian leukosis virus subgroup J (ALV-J), avian leukosis virus subgroup A (ALV-A), avian leukosis virus subgroup B (ALV-B), avian infectious bronchitis virus (IBV), Marek's disease virus (MDV), avian infectious laryngotracheitis virus (ILTV), Fowlpox virus (FPV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian reovirus (ARV), reticuloendotheliosis virus (REV), avian influenza virus (AIV) and Escherichia coli. The only specimens that yielded a strong signal were ALV-J, ALV-A and ALV-B, indicating that this assay is suitable for the detection of ALV. The limit of detection of this assay was 1.25 ng/ml of rp27 protein and 10(1.79)TCID(50) units of HLJ09MDJ-1 (ALV-J). Moreover, this AC-ELISA can detect ALV in cloacal swabs of chickens experimentally infected as early as 12 days post-infection. The AC-ELISA detected the virus in the albumin and cloacal swabs of naturally infected chickens, and the results were confirmed by PCR, indicating that the AC-ELISA was a suitable method for the detection of ALV. This test is rapid and sensitive and could be convenient for epidemiological studies and eradication programs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Nucleoprotein (NP) of influenza viruses is utilized to differentiate between the A, B, and C viral serotypes. The availability of influenza genome sequence data has allowed us to identify specific amino acids at particular positions in viral proteins, including NP, known as "signature residues," which can be used to discriminate human influenza A viruses from H5N1 highly pathogenic avian influenza in human cases (HPAI) and pandemic H1N1(2009) (H1N1/2009) viruses., Methods: Screening and epitope mapping of monoclonal antibodies (mAb) against NP of influenza A, which reacted differently with NP from human influenza A virus from HPAI and H1N1/2009 A virus. To identify the epitope(s) responsible for the discrimination of viral NP by mAbs, we prepared mutant NP proteins in the 293 cell expression system because some of the mAbs reacted with non-linear epitopes., Results and Conclusions: In the present study, we identified 3 mAbs. The results of epitope mapping showed that the epitopes were located at the signature residues. These results indicated that signature residues of NP could discriminate influenza A viruses from different origin., (© 2012 Blackwell Publishing Ltd.)

A new modified triple-antigen detection test was developed for the direct detection of the influenza A virus. The nucleoprotein (NP), matrix (M), and non-structural (NS1) proteins were used as target antigens because they are abundant in infected cells. Monoclonal antibodies specific to the NP, M, and NS1 proteins were generated. The antibody pairs were selected and evaluated for their reactivity individually and in combination in the triple-antigen detection using sandwich ELISA. Triple-antigen detection demonstrated a higher sensitivity than individual antigen detection when tested with both the H1N1 and H3N2 influenza A viruses. This was illustrated by the 4-fold lower limit of detection of the triple-antigen test than the individual antigen detection test. The findings demonstrated that the sensitivity of influenza A antigen detection was improved with the triple-antigen detection system as compared to individual antigen detection. Therefore, this technique could be a useful tool for the direct detection of cell-associated influenza A antigen. Furthermore, it could provide a basis for the development of a rapid triple-antigen test for influenza A diagnosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A simple gold nanoparticle (GNP) probe based assay (GNPA) that was modified from a bio-barcode assay (BCA) technique, was developed for ultra-sensitive, rapid detection of the bluetongue virus (BTV) VP7 outer-core protein. This assay captures the VP7 target antigen using the GNP probe coated with anti-VP7 polyclonal antibodies and single-stranded signal DNA. Magnetic microparticle (MMP) probes coated with anti-VP7 monoclonal antibodies were then added to form a sandwich immuno-complex. The single-stranded signal DNA coated onto the GNP probe present in the immuno-complex could then be detected by PCR and real-time fluorescence PCR using a TaqMan probe. The assay has a purified VP7 detection limit of 10(-2)fg/ml which is 8 orders of magnitude greater than that of conventional antigen capture ELISAs and 1 order of magnitude more sensitive than that of a conventional BCA. These results indicate that the GNPA is a highly sensitive method for easy detection of BTV proteins and that it can be modified as needed to measure the presence of other proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Japanese encephalitis virus(JEV), tick-borne encephalitis virus(TBEV), and eastern equine encephalitis virus (EEEV) can cause symptoms of encephalitis. Establishment of accurate and easy methods by which to detect these viruses is essential for the prevention and treatment of associated infectious diseases. Currently, there are still no multiple antigen detection methods available clinically. An ELISA-array, which detects multiple antigens, is easy to handle, and inexpensive, has enormous potential in pathogen detection. An ELISA-array method for the simultaneous detection of five encephalitis viruses was developed in this study. Seven monoclonal antibodies against five encephalitis-associated viruses were prepared and used for development of the ELISA-array. The ELISA-array assay is based on a "sandwich" ELISA format and consists of viral antibodies printed directly on 96-well microtiter plates, allowing for direct detection of 5 viruses. The developed ELISA-array proved to have similar specificity and higher sensitivity compared with the conventional ELISAs. This method was validated by different viral cultures and three chicken eggs inoculated with infected patient serum. The results demonstrated that the developed ELISA-array is sensitive and easy to use, which would have potential for clinical use.

Porcine astroviruses (PAstVs) are small RNA viruses associated with gastroenteritis. The capsid polyprotein of PAstVs, human (HAstVs) and feline (FAstVs) AstVs has a high similarity at the N-terminus before residue 415. Previous results showed the cross-detection of PAstVs and HAstVs from diarrheal samples using a commercial ELISA test that uses a monoclonal antibody to capture HAstVs, suggesting the existence of common immunoreactive epitopes between these two virus types. In this study, we seeked immunoreactive peptides located in the PAstV capsid that may be potentially used for their specific detection. The variability and hydrophobicity of a short fragment of 132 amino acids were analyzed using several capsid sequences of PAstVs and HAstVs. Peptides TATL, SLNP and IDIV were selected, synthesized and inoculated into rabbits. Pre- and hyperimmune sera were collected and their reactivity was examined by immunoassay and immunofluorescence against two wild-type strains of PAstV adapted to grow in cell culture, observing reactivity in two of the sera. Finally, the possible cross-reactivity of the sera against HAstVs was partially ruled out using HAstV8. Our data suggest that TATLGTIGSNSSGKTELEAC and IDIVVGKAATFNLKASDLSGP peptides represent immunoreactive regions useful for the specific detection of PAstVs., (Copyright © 2011 S. Karger AG, Basel.)

Japanese encephalitis virus (JEV) is a human pathogenic, mosquito-borne flavivirus that is endemic/epidemic in Asia. JEV is rarely detected or isolated from blood or cerebrospinal fluid (CSF), and detection of IgM is generally diagnostic of the infection. The flavivirus nonstructural glycoprotein NS1 is released transiently during flavivirus replication. The aim of this study was to set up a quantitative JEV NS1 antigen capture assay. A soluble hexameric form of JEV NS1 protein was produced in a stable Drosophila S2 cell clone and purified from supernatant fluids. Two IgG1 monoclonal antibodies (MAbs) with high affinity against two different epitopes of JEV NS1 antigen were used to develop an antigen-capture assay with a limit of detection of 0.2ngml(-1) NS1. Up to 1μgml(-1) JEV NS1 protein was released in supernatants of mammalian cells infected with JEV but <10ngml(-1) was released in sera of virus-infected mice before the onset of encephalitis and death. Moreover, NS1 protein was detected at low levels (<10ngml(-1)) in 23.8% of sera and in 10.5% of CSF of patients diagnosed as IgM-positive for JEV. This quantitative test of NS1 protein is proposed for highly specific diagnosis of acute infection with JEV genotypes I to IV., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The ultrasensitive bio-barcode amplification assay (BCA) technique was developed for the specific detection of the outer-core protein VP7 of bluetongue virus (BTV). The target antigen VP7 was first captured by gold nanoparticles (GNPs) coated with polyclonal antibodies. Magnetic microparticles (MMP) coated with VP7 monoclonal antibody were then added to form a sandwich immuno-complex. After the immuno-complex was formed, signal DNA annealed to DNA strands covalently bound to the GNPs were released by heating and characterized by PCR and real-time fluorescence PCR. A detection limit of 0.1fg/ml was measured for purified VP7, seven orders of magnitude more sensitive than that of conventional antigen capture ELISA. The BCA demonstrated the same enhanced sensitivity for detecting BTV in serum samples from sheep. In the following work it is demonstrated that this assay is a highly sensitive method for the detection of BTV proteins that could be adapted to measure other proteins., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Current methods for detection of avian influenza virus (AIV) based on virus culture and RT-PCR are well established, but they are either time consuming or require specialized laboratory facilities and highly trained technicians. A simple, rapid, robust, and reliable test, suitable for use in the field or at the patient's bedside, is urgently needed. In this study, the performance of a newly developed portable impedance biosensor was evaluated by comparison with real-time reverse transcriptase PCR (rRT-PCR) and virus culture for detection of AIV in tracheal and cloacal swab samples collected from experimentally H5N2 AIV infected chickens. The impedance biosensor system was based on a combination of magnetic nanobeads, which were coated with AIV subtype-specific antibody for capture (separation and concentration) of a target virus, and a microfluidic chip with an interdigitated array microelectrode for transfer and detection of target virus, and impedance measurement of the bio-nanobeads and AI virus complexes in a buffer solution. A comparison of results obtained from 59 swab samples using virus culture, impedance biosensor and rRT-PCR methods showed that the impedance biosensor technique was comparable in sensitivity and specificity to rRT-PCR. Detection time for the impedance biosensor is less than 1h., (Copyright © 2011 Elsevier B.V. All rights reserved.)

This paper describes the improvement of a rapid diagnostic test for the detection of rinderpest virus (RPV) at pen-side and the development of a similar test for the detection of another Morbillivirus, peste de petits ruminants virus (PPRV). Using the Svanova Biotech format, prototype chromatographic strip test devices were developed for RPV and PPRV detection. For the RP device, the incorporation of a monoclonal antibody (Mab), which recognises additional RPV strains of RPV lineage 2, enhanced the range of reactivity of the rapid diagnostic test. The device detected antigen in animals infected experimentally with different RPV strains. It also showed detection levels similar to the RP Clearview™ device reported previously. In addition, RPV was also detected under field conditions in Pakistan. A PPRV specific Mab (C77) was used for the development of the PPR test. This Mab recognised a wide range of PPRV isolates and did not show any cross-reactivity with any other virus tested. In animal experiments the device was able to detect viral antigen in eye swabs taken from the animals. The PPRV test should be invaluable for future PPR control eradication programs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A strip test for the dual detection of white spot syndrome virus (WSSV) and yellow head virus (YHV) was developed using monoclonal antibodies (MAbs) specific to the WSSV major envelope protein VP28 (W1 and W30) and the YHV nucleocapsid protein p20 (Y19 and Y21). The MAbs W30 and Y19 were conjugated with colloidal gold and sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. The MAbs W1 and Y21 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated W, Y and C, respectively. These test strips were placed in plastic cases and stored desiccated in a plastic bag. The test strips were assessed for their ability to detect WSSV and YHV simultaneously using pleopods sampled from shrimp. A pleopod homogenate in application buffer 100μl was applied to the sample chamber to flow through the nitrocellulose membrane strip, and antibody-protein complexes could be observed within 15min. In sample from shrimp infected with WSSV and/or YHV, viral protein bound to the colloidal gold-conjugated MAbs. These complexes were captured by the MAbs at the W and/or Y test lines, resulting in the appearance of reddish-purple coloured bands. Any unbound colloidal gold-conjugated MAbs migrated pass the W and Y lines would be captured by the GAM antibody, forming a band at position C. When samples not containing WSSV and YHV proteins or containing viral proteins at below the detection limit of the test, only the band at position C was observed. The sensitivity of the test was comparable to dot blot tests using single MAbs, and ∼500-fold less sensitive than a 1-step PCR test for WSSV and 1000-fold less sensitive than an RT-PCR test for YHV. Despite this lower sensitivity, the dual strip test has advantages in speed and simplicity in not requiring sophisticated equipment or specialized skills. The ability to co-detect WSSV and YHV provides simultaneously cost savings., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Dengue virus (DENV), a member of the Flavivirus family, has four distinct serotypes (DENV serotype 1 [DENV1], DENV2, DENV3, and DENV4) that require differentiation for the effective prevention of morbid disease. Early and rapid differentiation between flaviviruses remains challenging. Full assays combining four individual, serotype-specific and one group-specific nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies (MAbs) against DENV NS1 were developed and validated. The sensitivities and specificities of the full NS1 ELISAs were evaluated with viral cultures and dengue acute-phase sera. Four serotype-specific NS1 ELISAs displayed high specificities for the detection and differentiation of appropriate serotypes. The group-specific NS1 ELISA was broadly reactive with the four dengue virus serotypes. None of the NS1 ELISAs displayed cross-reactivity with the other flaviviruses or samples from febrile patients with non-dengue virus infections. The full serotype- and group-specific MAb-based NS1 capture ELISAs may provide tools for the early detection and typing of dengue infection, which is preferable to reverse transcriptase PCR (RT-PCR) for the rapid differential diagnosis of dengue virus infection in the field.

Here we describe a new analytical method for the detection of two influenza A viruses by nitrocellulose membrane and magnetic sensors that employ a special frequency mixing technique. The combination of the nitrocellulose membrane and magnetic bead detection permits a rapid assay procedure and excludes two steps (the development of color and the stop reaction) required for usual immunochemical detection methods such as ELISA. Quantitative virus detection was performed using magnetic beads conjugated with secondary antibody. The results were compared with conventional assay methods and with a dot-blot assay with fluorescence compound (FITC). Under optimum conditions, our new assay procedure is capable of detecting picograms of virus per well. This new method combining the nitrocellulose membrane and magnetic bead detection reduces analytical time and allows stable and repeatable analyses of samples in point-of-care applications., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A lateral flow immunoassay (LFI) was developed to identify and diagnose foot-and-mouth disease virus (FMDV) serotypes O, A and Asia 1. Antibodies obtained from rabbits and guinea pigs immunized with cell-culture-adapted virus strains (O/CHA/99, A/GS/LX/66, Asia 1/CHN/05) and suckling-mouse adapted virus strains (O/AV99(L), A/AV88(L), Asia 1/YNBS/58) were used as capture antibodies. The diagnostic kit included three immunochromatographic strips of types O, A and Asia 1, and the type-specific results were confirmed by color on the test lines of the three strips. The LFI was evaluated using epithelial and vesicular samples (n=396) prepared from current and historical field samples (provide by the National Foot-and-Mouth Disease Reference Laboratory of China at Lanzhou Veterinary Research Institute). Negative samples (n=95) were collected from healthy animals. The diagnostic sensitivity of the LFI for FMDV serotypes O, A and Asia 1 was 88.3% compared to 89.7% obtained by the reference method of indirect-sandwich ELISA. The sensitivity of the LFI for FMDV type Asia 1 was higher at 92.1% compared to 90.5% for the ELISA. The specificity of the LFI was 97.1% compared with 97.4%., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A monoclonal antibody (MAb) specific for the bluetongue virus (BTV) group specific antigen (VP7) was characterized for its reactivity with purified virus and recombinant BTV VP7 (rVP7) protein and its suitability for use in the sandwich ELISA. The MAb, designated as 5B5 was specific to VP7 and belongs to IgG2a subclass and was selected for the development of the sELISA in this study. The MAb had a titer of 1:25 with BTV and 1:2 with the rVP7 protein. The sELISA is based on capturing of BTV antigen with VP7 specific MAb followed by detection using BTV polyclonal antiserum raised in rabbits. The assay was evaluated with six cell culture adapted serotypes of BTV that have been isolated from India, 1, 2, 15, 17, 18 and 23. The assay could detect BTV antigen as early as day 8 in blood. It was also successfully applied for the detection of BTV group specific antigen in clinical samples of blood, washed RBCs, buffy coat and plasma. A total of 102 field samples from animals, suspected of being infected with BTV, were tested and 29.42% were positive. The blood samples were also amplified in cell culture which improved the sensitivity of the assay. Results confirmed that the sELISA is rapid and specific.

A nanoparticle-based immunoassay was developed for the rapid and sensitive detection of avian influenza virus (AIV). In this method, AIV-specific pentabody (pVHH3B) was conjugated to magnetic nanoparticles (MNPs) and used to capture AIV. Gold nanoparticles (GNPs), labelled with the anti-AIV mouse monoclonal antibody 3C8, were used as a detector. In the presence of target samples, the pentabody pVHH3B enriched AIV on the MNPs. Thereafter, mAb 3C8-labelled GNPs (GNPs-mAb3C8) bound to MNPs via AIV and were separated using a magnetic field. GNPs in the complex catalyzed the oxidation of hydroquinone to quinone, and the OD value of quinone was measured. The developed assay displayed substantial signal change after incubation in an AIV sample in a concentration-dependent manner. The detection limit was 10 ng/ml, which is 10 times more sensitive than conventional double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). In conclusion, by combining MNPs and a novel pentabody pVHH3B, this study provided a sensitive influenza viral detection assay that has the potential to become a rapid, sensitive and inexpensive diagnostic tool for infectious diseases., (Copyright © 2010. Published by Elsevier B.V.)

A polyclonal antibody-based antigen-capture ELISA (AC-ELISA) has been developed for detection of Canine parvovirus (CPV) antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1:1 600 and 1:400, respectively, in the check-board titration. In this study, a total of 152 samples (129 faecal samples and 23 cell culture supernatant) were tested both by AC-ELISA and by polymerase chain reaction (PCR). Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 10(2.8) TCID(50)/mL whereas PCR sensitivity was 10(0.8) TCID(50)/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.

Japanese encephalitis (JE) is caused by the Japanese encephalitis virus (JEV). It is a major public health problem in Asia. JEV infects swine which results in fatal encephalitis, abortion and stillbirth in pregnant sow, and hypospermia in boars. Swine is a viral amplifier, and thus plays a critical role in JEV transmission. Thus, development of a rapid method for JEV detection in swine is required for clinical JE diagnosis, as well as to suppress viral spread. In this study, a convenient and rapid immunochromatographic strip (ICS) was developed for detecting JEV in swine using two monoclonal antibodies (MAbs) (2A2 and 4D1) against the E protein of JEV. Results showed that colloidal gold-conjugated MAbs 2A2 (CG-MAb) bond with JEV and the resulting complex was held by the other MAb 4D1 at the test line to give a reddish-purple band. Sensitivity tests demonstrated that ICS can detect 2.5x10(5)PFU of JEV. The clinical screening results showed that the specificity and sensitivity of the ICS were 99.3% and 85.7% respectively as compared to that of RT-PCR. This suggests that the MAbs-based ICS test can be used as a convenient method for the rapid detection of JEV in infected swine samples., (Copyright 2010 Elsevier B.V. All rights reserved.)

A double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detection of the soft-shelled turtle iridovirus (STIV) was developed using a specific monoclonal antibody (mAb) against STIV and anti-STIV rabbit serum. Using DAS-ELISA, the detection limit of STIV was found to be 10(3)PFU/ml. The positive rate of 15 STIV samples was 100%, while the positive rate of 100 other aquatic virus samples was 0%. These data show that DAS-ELISA is highly specific and sensitive for the detection of STIV. In clinical tests, 128 samples isolated from pond-reared turtles were subjected to DAS-ELISA and PCR. The overall agreement between the results obtained by DAS-ELISA and PCR was 98.4%. The results indicate that the DAS-ELISA method could be used for diagnosing diseases caused by STIV., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Human herpesvirus-6 (HHV-6) infection, mostly caused by variant B, is common after transplantation. Here, we report a new modified method using an HHV-6B glycoprotein IgG antibody, OHV-3, and attempt to quantify the HHV-6 antigenemia after liver transplantation. Twenty-four liver transplant recipients were frequently monitored by the HHV-6 antigenemia test, which detects the HHV-6B virion protein in peripheral blood mononuclear cells (PBMC). HHV-6B antigens were now retrospectively demonstrated using a glycoprotein OHV-3 IgG antibody in the immunoperoxidase staining from the same specimens and quantified as positive cells/10,000 PBMC. The results were confirmed and quantified by DNA hybridization in situ. Altogether, 206 blood specimens were analyzed. During the first six months, HHV-6 antigenemia was detected in 17/24 (71%) recipients by using the HHV-6B virion antibody. In total, 37% (77/206) of specimens were positive with the virion antibody and 39% (78/201) by the OHV-3 antibody. The peak number of OHV-3-positive cells in the PBMC varied from 5 to 750/10,000 (mean 140/10,000). The OHV-3 antibody was useful to quantify the HHV-6B antigenemia. The findings of the HHV-6B quantitative antigenemia using the OHV-3 antibody correlated well with the previous qualitative HHV-6 antigenemia assay, and can be used as an alternative quantitative method in the monitoring of HHV-6 in transplant patients.

Influenza virus diagnosis has traditionally relied on virus isolation in chicken embryo or cell cultures. Many laboratories have adopted rapid molecular methods for detection of influenza viruses and discontinued routine utilization of the relatively slow viral culture methods. We describe an influenza A virus reporter cell line that contributes to more efficient viral detection in cell culture. Madin-Darby canine kidney (MDCK) cells were engineered to constitutively produce an influenza virus genome-like luciferase reporter RNA driven by the canine RNA polymerase I promoter. Induction of a high level of luciferase activity was detected in the Luc9.1 cells upon infection with various strains of influenza A virus, including 2009 H1N1 pandemic and highly pathogenic H5N1 virus. In contrast, infection with influenza B virus or human adenovirus type 5 did not induce significant levels of reporter expression. The reporter Luc9.1 cells were evaluated in neutralizing antibody assays with convalescent H3N2 ferret serum, yielding a neutralization titer comparable to that obtained by the conventional microneutralization assay, suggesting that the use of the reporter cell line might simplify neutralization assays by facilitating the establishment of infectious virus endpoints. Luc9.1 cells were also used to determine the susceptibility of influenza A viruses to a model antiviral drug. The equivalence to conventional antiviral assay results indicated that the Luc9.1 cells could provide an alternative cell-based platform for high-throughput drug discovery screens. In summary, the MDCK-derived Luc9.1 reporter cell line is highly permissive for influenza A virus replication and provides a very specific and sensitive approach for simultaneous detection and isolation of influenza A viruses as well as functional evaluation of antibodies and antiviral molecules.

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10(4) PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r(2) value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r(2) value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.

Objective: Culture-confirmation of rhinovirus is done using acid lability testing, a laborious and time-consuming method which delays the reporting of patient results by 1-2 days. A fluorescent monoclonal antibody pool (Light Diagnostics Pan-Enterovirus Blend; Millipore Inc., Temecula, Calif., USA) developed to identify various enterovirus isolates in culture was recently reported to also cross-react with rhinoviruses. We evaluated the use of this cross-reacting antibody, used in tandem with non-cross-reacting enterovirus antibodies (D(3) IFA; Diagnostic Hybrids Inc., Athens, Ohio, USA) to rapidly identify rhinoviruses in cell culture., Methods: Microscope slides were prepared from cell cultures of 11 rhinovirus clinical isolates and a variety of other respiratory viruses. Slides were stained using both enterovirus antibody pools and examined for fluorescent activity., Results: Positive fluorescence was observed in all the rhinovirus isolates tested using the pan-enterovirus antibody blend, but yielded negative results when stained using the D(3) antibodies. Both antibody products produced positive fluorescence for enterovirus isolates and produced negative results when a variety of other respiratory viruses were examined., Conclusion: Staining suspected rhinovirus isolates with each antibody pool affords a rapid means of identifying rhinoviruses and distinguishing them from enteroviruses, without the need for acid lability testing., (Copyright 2010 S. Karger AG, Basel.)

Human papillomavirus (HPV) L1 self-assembles into virus-like particles (VLPs), which are the basis for the two commercially available prophylactic vaccines. For one of them (Cervarix) HPV 16 and 18 VLPs are being produced in insect cells using the baculovirus expression system. However, due to low yield, production of VLPs remains challenging for certain other PV types. Here we report that employment of a modified baculovirus-based (MultiBac) expression system (Berger, I., Fitzgerald, D. J., and Richmond, T. J. (2004). Baculovirus expression system for heterologous multiprotein complexes. Nat. Biotechnol. 22(12), 1583-7) permits substantially improved VLP production of several PV types up to 40-fold. Highest VLP yields were achieved when two copies of the L1 gene were expressed from independently controlled cassettes. We have evaluated the production of HPV 57 L1 VLPs by the MultiBac system in more detail. Whereas the level of the HPV 57 L1 protein was only slightly increased in comparison to the standard protocol we monitored a strongly enhanced yield of HPV 57 VLPs. Our results imply that a critical concentration of L1 within the producer cell is required for efficient VLPs assembly. We show evidence that in addition a dominant negative factor in conventionally produced recombinant baculoviruses contributes to differences in VLP yield. This phenomenon might be attributable to the absence of the viral cysteine protease V-CATH in the modified baculovirus system. We anticipate that use of the MultiBac expression system will facilitate capsid production for papillomaviruses and thereby enable the generation of vaccines against infections by many of the as yet untargeted HPV types.

We report the application of an integrated optical Young interferometer sensor for ultrasensitive, real-time, direct detection of viruses. We have validated the sensor by detecting herpes simplex virus type 1 (HSV-1), but the principle is generally applicable. Detection of HSV-1 virus particles was performed by applying the virus sample onto a sensor surface coated with a specific antibody against HSV-1. The performance of the sensor was tested by monitoring virus samples at clinically relevant concentrations. We show that the Young interferometer sensor can specifically and sensitively detect HSV-1 at very low concentrations (850 particles/mL). We have further demonstrated that the sensor can specifically detect HSV-1 suspended in serum. Extrapolation of the results indicates that the sensitivity of the sensor approaches the detection of a single virus particle binding, yielding a sensor of unprecedented sensitivity with wide applications for viral diagnostics.

The rapid detection of influenza viruses is important for forming preventative strategies, directing initiation of anti-viral therapy, detecting potential avian influenza viruses, and excluding influenza-like pathogens, such as SARS. The ImmunoCard STAT! Flu A and B Plus test (Meridian Bioscience, Cincinnati, OH) is a new point of care (POC) test utilizing influenza-specific monoclonal antibodies for rapid diagnosis. The performance of this assay was compared to the established POC Binax NowFlu A and NowFlu B test, and the reference diagnostic standards of viral culture, indirect immunofluorescence (IFA), and RT-PCR where appropriate. Testing of nasopharyngeal aspirates (NPA) from children, throat swabs, and nasal swabs from adults indicated ImmunoCard STAT! specificity of 98% and 100% for influenza A and B, respectively in 224 specimens. The Binax test showed specificity of 99% and 100%, respectively for influenza A and B. Sensitivity results were identical for both rapid detection kits (80% and 47% for Flu A and B, respectively). Overall results were very similar for both testing devices with the advantage of ImmunoCard STAT! Flu A and B Plus test detecting influenza A and B with sharp and easy to read results., (Copyright 2006 Wiley-Liss, Inc.)

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.

Aims: Rapid detection and quantification of viruses is crucial in clinical practice, veterinary medicine, agriculture, basic research as well as in biotechnological factories. However, although various techniques were described and are currently used, development of more rapid, more sensitive and quantitative methods seems to be still important., Methods and Results: Here we describe a method for rapid detection of viruses (using bacteriophages as model viruses), based on electrical biochip array technology with the use of antibodies against capsid proteins., Conclusions: Using the procedure developed in this work, we were able to detect 2 x 10(4) virions on the chip. The whole assay procedure takes c. 50 min and the assay is quantitative., Significance and Impact of the Study: This procedure may be useful in various approaches, including detection of bacteriophage contamination in bioreactors and possibly detection of toxin gene-bearing phages or other viruses in food samples.

A simple, sensitive, specific and rapid procedure for isolating and typing polioviruses is described. Specimens are inoculated onto confluent monolayers of cell lines (Hep-2C, L20B or RD) seeded into microtitre plates. After 24-48 h, the infected cells are stained with monoclonal antibodies specific for poliovirus types 1,2,3 or a blend of the three antibodies followed by an anti-mouse IgG-horseradish peroxidase conjugate. On addition of substrate, infected cells stain an intense blue colour and are easily distinguished from uninfected cells by light microscopy. Poliovirus infection can be detected before the appearance of cytopathic effects (CPE). This Blue-Cell ELISA test was evaluated against conventional culture and seroneutralisation on a range of polio isolates and clinical specimens. The sensitivity and specificity of the Blue-Cell ELISA compared to neutralisation was 100% (87/87) on culture supernatants of poliovirus isolates sent to our reference laboratory for confirmation. All the poliovirus isolates were typed within 24 h of specimen inoculation using the new method compared to 6-10 days by conventional culture and neutralisation. The method proved to be more sensitive than conventional culture when clinical specimens were examined. Of 43 clinical specimens from which poliovirus had been previously isolated by various laboratories in the U.K., 30/43 (69.8%) were positive for poliovirus by the Blue-Cell ELISA compared to 29/43 (67.4%) by conventional culture and neutralisation. Neutralisation of specimens exhibiting CPE indicated that all of the polioviruses were correctly typed with the new method. CPE was not observed by conventional culture in any specimen that was negative in the Blue-Cell ELISA. There were no cross-reactions with a range of other enteroviruses.

Th baculovirus expression system is a system of choice for expressing eukaryotic proteins. Large amounts of biologically active material can be generated using this system by infecting insect cells with a baculovirus expressing the target protein. At several stages during the production of a baculovirus stock, it is necessary to titer the virus. Current methods have long time lines and are either technically difficult or are limited to viruses expressing a reporter gene. The new assay described here yields titers in 48 h, is easy to perform using 96-well plates, and is applicable to any Autographa californica nucleopolyhedrovirus-based recombinant baculovirus. This assay uses an antibody to a viral envelope glycoprotein to detect infected cells via immunostaining. The titer is determined by counting foci of infection under a light microscope. The required incubation period is shortened considerably because infected cells express viral antigens long before the macroscopic signs of infection scored in other assays become apparent. Titers determined using this immunological assay are comparable, both in value and variability, to those obtained using a traditional method, provided that the stocks have titers above 10(4) pfu/ml., (Copyright 1999 Academic Press.)

The Digene Hybrid Capture System (DHCS) is a solution hybridization antibody capture assay for the chemiluminescent detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. This assay was compared with the CMV antigenemia assay and shell vial and tube cultures for the detection of CMV in 234 blood specimens from 72 patients with human immunodeficiency virus. Intra- and interrun precision of the DHCS assay gave coefficients of variation of 17.8 and 16.3%, respectively. The correlation coefficient for the quantitative results obtained by the DHCS assay and the antigenemia assay was 0.911 (95% confidence interval, 0.885 to 0.930). Agreement between the DHCS assay and the other three assays ranged from 83 to 86%. The DHCS assay detected 71, 87, and 84% of specimens that were positive by antigenemia, shell vial cultures, and tube culture, respectively. A total of 92% of specimens that were positive by the DHCS assay were also positive by at least one of the other assays. Evaluation of the usefulness of quantitation of CMV DNA by using the DHCS assay and its correlation with clinical disease demonstrated that, with some exceptions, patients with clinical CMV disease tended to have high levels of DNA whereas asymptomatic patients tended to have low or undetectable levels. Overall, the DHCS assay provided a rapid, quantitative, and objective measure of CMV activity in leukocytes, but results did not always correlate with clinical disease.

To assess the risks from viral contamination of drinking-water supplies, there is a clear need for methods to directly detect viral pathogens. In this study, we developed a broad-spectrum immunocapture method for concentration and purification of enteric viruses. The method involved indirect antibody capture (AbCap) of intact viruses followed by release of virion genomic RNA and reverse transcriptase PCR for amplification and oligoprobe hybridization for detection. The procedure involved concentrating enteric viruses from large volumes of water by standard filtration-elution techniques with IMDS filters and 1 liter of 1% beef extract-0.05 M glycine (BE/G) as an eluate. The BE/G eluate was concentrated and purified by polyethylene glycol (PEG) precipitation, Pro-Cipitate (a commercially available protein precipitating reagent) precipitation, and a second PEG precipitation to a volume of approximately 500 mu l. Aliquots of the second PEG precipitate were further processed by RNA extraction, AbCap, or cell culture analysis for infectious viruses. The AbCap method was applied to 11 field samples of fecally contaminated surface water. Of the 11 samples, 9 were positive for enteric viruses by AbCap method 4 of 11 samples were positive for enteric viruses by direct RNA extraction of a small aliquot of the second PEG concentrate; and 4 of 11 samples were positive for enteric viruses by measurement of cell culture infectivity. The results of enteric viruses were compared with those for standard bacterial and coliphage indicators of fecal contamination.

Conventional tube cell culture was compared with a 72-h, spin-amplified shell vial indirect immunofluorescence assay for the detection of enterovirus from clinical specimens. The sensitivity for the shell vial assay after resolution of discrepant results were 93 and 100%, respectively. The shell vial assay detected 93% of the positive cultures within 72 h of incubation while conventional tube culture detected only 51% of the positive cultures within the same time interval. The data suggest that a spin-amplified shell vial indirect immunofluorescence assay may be useful for the detection of enterovirus from clinical specimens.

Enzyme-linked immunosorbent assay (ELISA) enabled a more precise serological typing of isolates of red clover necrotic mosaic virus (RCNMV) originating from Slovak and Czech Republics, Poland, Sweden and Great Britain. We found 5 isolates of serotype A, 14 isolates of serotype B, and 6 isolates of serotype C. Some isolates represented mixtures of serotypes, namely A+B (1 isolate), A+C (1), B+C (7), and A+B+C (5). ELISA was found to be a more suitable method of serotype identification of RCNMV isolates than the double diffusion agar gel test for its higher sensitivity and greater selectivity.

A new technique was developed for visualizing virus particles electrophoretically transferred from infected leaves to nitrocellulose membranes. This technique was used to study the distribution of peanut mottle virus (PMV) particles in vivo. The immobilized transferable virus proteins from leaf tissue to nitrocellulose membranes were detected by an enzyme-linked immunobinding technique. The free binding groups on the nitrocellulose membrane were blocked with casein. The nitrocellulose membrane was then treated with PMV-specific antiserum. The virus-bound antibodies were located by protein A-peroxidase or protein A-alkaline phosphatase conjugate followed by the peroxidase substrate mixture, 4-chloro-1-naphthol and H2O2, in the first case or the alkaline phosphatase substrate mixture, 5-bromo-4-chloro-3-indolyl phosphate and Nitroblue tetrazolium in the second case. The locations of virus particles were detected by the corresponding reaction product. A mirror image was obtained on nitrocellulose membrane showing a pattern identical to that of necrotic lesions on leaf pieces but with a significantly larger size of local lesion copy, indicating the presence of virus particles in the apparently healthy tissue outside the necrotic lesion. The new technique is expected, because of its relative simplicity and sensitivity, to be useful for many other fields of biological research where the in vivo distribution of pathogens, antigenic molecules including drugs, or cells of specific antigenic molecules such as cancer or genetically engineered cells needs to be investigated.

A monoclonal antibody was made which identifies a 160-180 kDa structural protein in guinea pig cytomegalovirus (GPCMV) infected cells by Western blot using non-reducing conditions. This protein was shown to be a virion structural protein by purification of GPCMV on a density viscosity gradient and Western blot analysis. Phosphoanacetic acid (PAA) experiments suggest that the protein is a late GPCMV protein. In vitro the monoclonal antibody labels a cytoplasmic protein in infected guinea pig embryo fibroblasts by 12 h postinfection. The monoclonal antibody also identifies GPCMV infected cells in vivo in paraffin embedded formalin fixed tissue.

Rotavirus (RV) is known to be the most common cause of severe diarrhoea in infants and young children, each year leading to an estimated 800,000-900,000 deaths. RV also infects bovines and other species, with high morbidity and mortality. A rapid and simple 'naked-eye' dipstick system was developed to detect human RV in faeces, using nitrocellulose as solid phase, two monoclonal antibodies, and colloidal gold as marker. The system detects 10(4) viral particles (1-2 ng)/g of faeces. For human RV the specificity and sensitivity were 100% when compared with a commercial latex system, and 99% and 98%, respectively, when correlated with traditional RNA-PAGE, and 100% and 98% compared to an ELISA system.

A method for the detection of RNA viral and subviral plant pathogens was developed that combines pathogen partial purification by solid-phase adsorbed antibodies, reverse transcriptional-polymerase chain reaction (RT-PCR) and quantitation of the amplified products by fluorescence. The reverse transcription of the RNA is performed directly on the retained material without any previous thermal or chemical disruption of the virus particles. The whole procedure can be carried out in a microtiter plate. Its validity has been successfully confirmed for the detection of bean yellow mosaic virus, cherry leafroll virus, cucumber mosaic virus, citrus tristeza virus, grapevine fanleaf virus, potato leafroll virus, pepper mild mottle virus, and tomato spotted wilt virus, as well as the satellite RNA of cucumber mosaic virus and potato spindle tuber viroid. In this procedure virus-specific antibodies can be replaced by monoclonal antibodies against double-stranded RNA, thus offering the possibility of detection when no specific virus antibodies are available, or immunological methods are difficult to use (i.e., subviral pathogens like satellite-RNAs or viroids). The method described has the typical sensitivity of assays based on the polymerase chain reaction, it is not more laborious than ELISA, and an equivalent degree of automation is possible.

Epizootic haematopoietic necrosis virus (EHNV) was grown in Bluegill fry (BF-2) cells and purified using differential and gradient centrifugation. The lower band (B2) from 15-60% sucrose gradients contained infective EHNV but few contaminating cell components when assessed by electron microscopy, SDS-PAGE, and Western blotting using anti-BF-2 serum and anti-B2 serum. Both rabbit and sheep anti-B2 sera precipitated B2 in agarose gel immunodiffusion and detected EHNV in cell culture supernatant when used in an indirect antigen-capture ELISA. Rabbit anti-B2 serum was used as capture antibody while sheep anti-B2 serum was used to detect viral antigen. Pre-adsorption of diluted sheep anti-B2 serum using BF-2 cell lysate greatly improved the specificity and sensitivity of the technique.

Quality control in diagnostic virology is in its infancy and some preliminary investigations are reported. Simulated specimens containing viruses have been prepared and distributed so that they could be included in the routine work of virus isolation in participating laboratories. Sera containing antibodies to rubella and influenza virus have also been sent to laboratories for testing. Laboratories were able to check their results immediately after the closing date, and could compare their performance with that of other laboratories when the analysis of each distribution was circulated. The results so far show considerable variation in performance and indicate that there is room for improvement in some laboratories. There is no evidence to show whether the exercise has had any effect on the practice of virology in participating laboratories but some baselines have been produced for comparison with the results of investigations in the future.

Intravenous injection of cells and their tissue culture supernatants (CS) from human lymphoblastoid cell lines (LCL) induced the formation of lesions on the chorioallantoic membrane (CAM) of the chicken embryo. Injection of cells and CS from non-LCL and normal human lymphocytes induced few or no lesions. Irradiated chick embryos were more sensitive to lesion formation than were nonirradiated embryos. The log10 CAM lesions induced in irradiated (500 rads) embryos were a linear function of the log10 cells (from LCL) in the inoculum; the slope was 1.0, within experimental error. The formation of CAM lesions did not depend on the presence of Epstein-Barr virus (EBV) since lesions were also induced by cells and extracts derived from EBV genome-free LCL. Lesion-inducing activity associated with CS was filterable through 0.22-mu filters, sedimented at 78,000 x g, and sensitive to inactivation by heat (56 degrees C for 30 min), UV irradiation, chloroform, sera from chickens immunized against CS, and certain human sera. Lesion-inducing activity associated with cells and extracts was resistant to 5,000 rads of gamma-radiation. B2/B2 embryos (the B locus is the major histocompatibility locus of chickens) were more sensitive to lesion formation than were B15/B21 and outbred embryos; this suggested a genetic influence on lesion formation. Our data suggest that the irradiated chicken embryo may be a highly sensitive and useful means for the detection of an unidentified or unknown agent or agents that may play an important role in human oncogenic lymphocyte transformation or might interact with transforming viruses.