End-point Dilution Method Research Articles

Anti-Coronavirus Activity of Chitosan-Stabilized Liposomal Nanocarriers Loaded with Natural Extracts from Bulgarian Flora.

Disease's severity, mortality rates, and common failures to achieve clinical improvement during the unprecedented COVID-19 pandemic exposed the emergency need for new antiviral therapeutics with higher efficacy and fewer adverse effects. This study explores the potential to encapsulate multi-component plant extracts in liposomes as optimized delivery systems and to verify if they exert inhibitory effects against human seasonal betacoronavirus OC43 (HCoV-OC43) in vitro. The selection of Sambucus nigra, Potentilla reptans, Allium sativum, Aesculus hippocastanum, and Glycyrrhiza glabra L. plant extracts was based on their established pharmacological and antiviral properties. The physicochemical characterization of extract-loaded liposomes was conducted by DLS and electrokinetics. Encapsulated amounts of the extract were evaluated based on the total flavonoid content (TFC) and total polyphenol content (TPC) by colorimetric methods. The BALB 3T3 neutral red uptake (NRU) phototoxicity/cytotoxicity assay was used to estimate compounds' safety. Photo irritation factors (PIFs) of the liposomes containing extracts were <2 which assigned them as non-phototoxic substances. The antiviral capacities of liposomes containing medicinal plant extracts against HCoV-OC43 were measured by the cytopathic effect inhibition test in susceptible HCT-8 cells. The antiviral activity increased by several times compared to "naked" extracts' activity reported previously. A. hippocastanum extract showed 16 times higher inhibitory properties reaching a selectivity index (SI) of 58.96. Virucidal and virus-adsorption effects were investigated using the endpoint dilution method and ∆lgs comparison with infected and untreated controls. The results confirmed that nanoparticles do not directly affect the viral surface or cell membrane, but only serve as carriers of the active substances and the observed protection is due solely to the intracellular action of the extracts.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Remdesivir-Loaded Nanoliposomes Stabilized by Chitosan/Hyaluronic Acid Film with a Potential Application in the Treatment of Coronavirus Infection.

An object of the present study was the development of liposomes loaded with the medicine Veklury® (remdesivir) stabilized by electrostatic adsorption of polysaccharide film formed from chitosans with different physicochemical characteristics and hyaluronic acid. The functionalization of the structures was achieved through the inclusion of an aptamer (oligonucleotide sequence) with specific affinity to the spike protein of the human coronavirus HCoV-OC43. The hydrodynamic size, electrokinetic potential and stability of the structures were evaluated at each step in the procedure. The encapsulation efficiency and loaded amount of remdesivir (99% and 299 µg/mL) were estimated by UV-vis spectroscopy. Our investigations showed manifestation of promising tendencies for prolonged periods of the drug release and increased effectiveness of its antiviral action. Among all studied versions of the delivery system, the most distinguished and suitable in a model coronavirus therapy are the liposomes formed from chitosan oligosaccharides. The cytotoxicity of the liposomes was determined against the HCT-8 cell line. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the compounds. The virucidal activity and the effect on the viral adsorption of the samples were reported by the end-point dilution method, and the alteration in viral titer was determined as Δlgs compared to untreated controls. The redox-modulating properties of the nanoparticles were studied in vitro in certain/several/a few chemical model systems. Our investigations showed a manifestation of promising tendencies for a prolonged effect of the drug release and increased effectiveness of its antiviral action.

To reduce cytotoxicity when testing the virucidal activity of chemical disinfectants and biocides: The “T-25 method” as an alternative to “large-volume-plating”

When testing the virucidal activity of biocides, the non-inactivated residual virus is titrated on cell cultures by the end point dilution method on 96-well tissue culture plates. However, residues of the biocide to be tested also come into contact with the cell cultures in varying concentrations and thus can lead to cytotoxic effects even at high levels of dilution. In the European standards for testing biocides, in particular disinfectants, methods such as Large-Volume-Plating (LVP) method and, in some guidelines, gel filtration procedures are described for reducing cytotoxic effects in the case of highly cytotoxic products, if the classical dilution method proves to be impractical.In order to enable the testing of highly cytotoxic biocides for their activity against viruses, an alternative method for reducing cytotoxicity is introduced, which is based on a procedure of isolating infectious viruses from cytotoxic patients’ materials such as stool and can be applied when the other methods fail.

Anti-Coronavirus Efficiency and Redox-Modulating Capacity of Polyphenol-Rich Extracts from Traditional Bulgarian Medicinal Plants

Background: The use of various herbal therapists as part of traditional medicine in different parts of the world, including Bulgaria, is due to the knowledge accumulated over the centuries by people about their valuable biological activities. In this study, we investigate extracts from widely used Bulgarian medicinal plants for their ability to prevent the coronavirus infection of cells by testing different mechanisms of antiviral protection, their polyphenol content, and redox-modulating capacity. Methods: The influence on the stage of viral adsorption, the inhibition of extracellular virions, and the protective effect on uninfected cells of the plant’s extracts were reported by the end-point dilution method, and virus titer (in Δ lgs) was determined as compared to the untreated controls. The total content of polyphenols and flavonoids was also determined. We tested the antioxidant power of the extracts by their ability to inhibit the generation of superoxide anionic radicals and to scavenge DPPH radicals. We determined their iron-reducing, copper-reducing, and metal-chelating antioxidant powers. Results: Most of the extracts tested suppress the extracellular virions of HCov. They also inhibit the stage of viral adsorption to the host cell to varying degrees and have a protective effect on healthy cells before being subjected to viral invasion. The examined extracts contained significant levels of polyphenols and quercetin-like flavonoids and showed remarkable antioxidant, radical, and redox-modulating effects. Conclusions: All of these 13 extracts from Bulgarian medicinal plants tested can act as antioxidants and antiviral and symptomatic drugs for the management of coronavirus infection.

NDV as an Oncolytic Agent - Study in Cancer Cell Lines

Newcastle disease virus (NDV) exhibits oncolysis in its natural form. This oncolytic virus (OV) has the potential to specifically infect, propagate, and lyse cancer cells while sparing the normal cells. This study was aimed to screen for oncolytic NDV strain isolated from poultry. A total of ten velogenic NDV strains were propagated in 10 day old embryonated SPF chicken eggs and allantoic fluid of these infected eggs was collected for further study. The virus enumeration was carried out by hemagglutination assay (HA) and end point dilution method. The cytopathic effect of ten NDV strains on cancer cell lines like MDA-MB-231, MCF-7, PC3, and A549 along with normal control cell line HEK293 was determined by MTT assay 72 hours post infection. These cell lines were infected with three doses (1, 0.1, and 0.01 MOI). DNA laddering effect of the screened NDV isolate was studied after infecting all cancer and normal cells at MOI 1. Morphological changes in MDA-MB-231 on infection with the screened NDV isolate were analyzed using H&amp;E hematoxylin and eosin staining. The screened NDV isolate showed the maximum cytopathic effect i.e. 61.55% on MDA-MB-231 at MOI 1 but had no potent cytotoxic effect on HEK293. DNA laddering effect was observed which confirmed the mode of death to be apoptosis. All the observed morphological changes in MDA-MB-231 were typical of the cytopathogenic effects of NDV on cancer cell lines. In conclusion, the screened oncolytic NDV shows effective oncolysis against MDA-MB-231 cell line. However, further study is required to determine the exact mode of action involved.

Bulgarian Medicinal Extracts as Natural Inhibitors with Antiviral and Antibacterial Activity.

Background: Bulgaria is a country with a wide range of medicinal plants, with uses in traditional medicine dating back for centuries. Methods: Disc diffusion assay was used to evaluate the antimicrobial activity of the plant extracts. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the extracts. The virucidal activity of the extracts, their influence on the stage of viral adsorption, and their protective effect on uninfected cells were reported using the end-point dilution method, and Δlgs was determined as compared to the untreated controls. Results: The results of the study reveal that the antibacterial potential of G. glabra and H. perforatum extracts in Gram-positive bacteria is more effective than in Gram-negative bacteria. When applied during the replication of HSV-1 and HCov-OC-43, only some of the extracts showed weak activity, with SI between 2 to 8.5. Almost all tested extracts inhibited the extracellular virions of the studied enveloped viruses (HSV-1 and HCov-OC-43) to a greater extent than of the non-enveloped viruses (PV-1 and HAdV-5). They inhibited the stage of viral adsorption (HSV-1) in the host cell (MDBK) to varying degrees and showed a protective effect on healthy cells (MDBK) before they were subjected to viral invasion (HSV-1). Conclusion: The antipathogenic potential of extracts of H. perforatum and G. glabra suggests their effectiveness as antimicrobial agents. All 13 extracts of the Bulgarian medicinal plants studied can be used to reduce viral yield in a wide range of viral infections.

Molecular investigation of proanthocyanidin from Alpinia zerumbet against the influenza A virus

Proanthocyanidins (PACs) have various bioactivities, such as being anti-bacterial, anti-cancer, and anti-oxidant. Consequently, they have been vigorously studied for the development of new natural bioactive compounds. Recently, PAC was isolated from leaves and pseudostems of the medicinal plant Alpinia zerumbet (Pers.) B.L. Burtt and R.M. Smith, and it had shown in vitro antiviral activity against influenza A H1N1 viruses (IAVs). The 50% endpoint dilution method indicated that 0.1 mg/mL A. zerumbet-derived PAC (AzPAC) reduced the titer of IAVs by >3 logs. The antiviral activity of AzPAC means that it can interact directly with viral particles since the antiviral activity test was done by coincubation of PAC with and IAVs before viral infection. However, few studies have investigated the preventive mechanism utilized by AzPAC on influenza virus replication. In this study, the composition of AzPAC and the affinity between AzPAC and IAVs was investigated in detail. We found that AzPAC was composed of an epicatechin, which was linked by inter-flavan bonds between the C4 and C8 positions (B2-type) and the C4 and C6 positions (B5-type) in the terminal units. A quenching assay indicated that AzPAC interacted with IAV membrane proteins, hemagglutinin and neuraminidase. Additionally, circular dichroism analysis indicated that AzPAC affected the change in the secondary structure rate of the viral membrane proteins. AzPAC was able to impair the infective process of IAVs via direct interaction with their viral membrane proteins. These results indicate that A. zerumbet is a bioresource for the development of preventive drugs against IAV infection.

Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID50) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).

Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected for the dilution), there can be issues with sensitivity and reproducibility, especially when adenovirus serotypes are used that infect standard cell lines with poor efficiency. An alternative approach is to plate serial dilutions of the cells growing in the wells of a 96-well tissue culture plate and determine the dilution at which 50% of the wells are infected. This ancient and reliable technique known as the "tissue culture infection dose 50%" (TCID50) end-point dilution method has been used for titering a number of viruses, especially those that do not readily form plaques. Usually, infected wells are determined by direct examination for cytopathic effect (CPE) or cell viability. However, by combining a 96-well TCID50 format and the power of quantitative polymerase chain reaction (qPCR) for detection, a large increase in sensitivity-in our hands 10-fold, with a range of both transgenes and adenovirus serotypes-can be achieved. This protocol uses a 96-well TCID50 format, in conjunction with qPCR for sensitive and quantitative positive-well calling, to determine infectious titer of adenovirus vectors.

Quantitative single dilution liquid phase blocking ELISA for sero-monitoring of foot-and-mouth disease in India

Three of the seven serotypes of foot-and-mouth disease (FMD) virus are prevailing in India. A massive vaccination campaign is on to control and eradicate the disease from the country. However, FMD vaccines provide short term immunity, hence regular assessment of antibody level in the vaccinated herds is indispensible for the success of the control programme. The antibodies are quantitatively estimated, either by virus neutralization test or by end-point dilution liquid-phase-blocking ELISA (LPBE). Millions of cattle and buffalo in the country are now systematically vaccinated, and thousands of serum samples are routinely screened in the country for estimation of herd immunity against FMDV serotypes O, A and Asia1. Testing such a large number of serum samples within limited a period of time by the conventional end point dilution method of LPBE requires lots of man power, and biological reagents. A more economical high throughput single dilution LPBE (SdLPBE) assay was optimized and validated for quantitative estimation of antibody levels against the three FMD virus serotypes. The assay was thoroughly validated against LPBE method before adopting it for country-wide use. The biological reagents used in the assay were prepared in thermo-stable form to enable transportation to the field level FMD diagnostic laboratories.

Screening and characterization of resistance to succinate dehydrogenase inhibitors in Alternaria solani

Field isolates of Alternaria solani, which causes early blight of potato in Idaho, USA were evaluated in vitro for their sensitivity towards the succinate dehydrogenase inhibitor (SDHI) fungicides boscalid, fluopyram and penthiopyrad. A total of 20 isolates were collected from foliar‐infected tissue in 2009, 26 in 2010 and 49 in 2011. Fungicide sensitivity was tested using the spiral‐gradient end point dilution method. The frequency of boscalid‐resistant isolates (&gt;50% relative growth when using a spiral dilution gradient starting at 507 mg L−1) drastically increased over the duration of this study (15% in 2009, 62% in 2010 and 80% in 2011). Increasing resistance to fluopyram and penthiopyrad was observed. However, cross‐resistance was only observed between boscalid and penthiopyrad. The target site of this fungicide class is the succinate dehydrogenase (SDH) enzyme complex, which is vital for fungal respiration. Sequence analysis of the SDH complex revealed mutations in the subunits B and D that were correlated with the emergence of boscalid resistance in potato fields in Idaho. In particular, H277R and H133R were identified in SDH subunits B and D, respectively. The presence of restriction sites in the gene sequences allowed the development of a rapid PCR‐RFLP method to assess boscalid sensitivity in A. solani populations.

High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization

BackgroundBudded baculoviruses are utilized for vaccine, the production of antibody and functional analysis of transmembrane proteins. In this study, we tried to produce and purify the recombinant Bombyx mori nucleopolyhedrovirus (rBmNPV-hPRR) that displayed human (pro)renin receptor (hPRR) connected with FLAG peptide sequence on its own surface. These particles were used for further binding analysis of hPRR to human prorenin. The rBmNPV-hPRR was produced in silkworm larvae and purified from its hemolymph using size exclusion chromatography (SEC).ResultsA rapid method of BmNPV titer determination in hemolymph was performed using quantitative real-time PCR (Q-PCR). A correlation coefficient of BmNPV determination between end-point dilution and Q-PCR methods was found to be 0.99. rBmNPV-hPRR bacmid-injected silkworm larvae produced recombinant baculovirus of 1.31 × 108 plaque forming unit (pfu) in hemolymph, which was 2.8 × 104 times higher than transfection solution in Bm5 cells. Its purification yield by Sephacryl S-1000 SF column chromatography was 264 fold from larval hemolymph at 4 days post-injection (p.i.), but 35 or 39 fold at 4.5 or 5 days p.i., respectively. Protein patterns of rBmNPV-hPRR purified at 4 and 5 days were the same and ratio of envelope proteins (76, 45 and 35 kDa) to VP39, one of nucleocapsid proteins, increased at 5 days p.i. hPRR was detected in only purified rBmNPV-hPRR at 5 days p.i..ConclusionThe successful purification of rBmNPV-hPRR indicates that baculovirus production using silkworm larvae and its purification from hemolymph by Sephacryl S-1000 SF column chromatography can provide an economical approach in obtaining the purified BmNPV stocks with high titer for large-scale production of hPRR. Also, it can be utilized for further binding analysis and screening of inhibitors of hPRR.

Development and characterization of a standardized ELISA including a reference serum on each plate to detect antibodies induced by experimental malaria vaccines

Enzyme linked immunosorbent assay (ELISA) has been widely used to measure antibody titers for evaluating the immunogenicity of a vaccine. However, there is as yet no generally accepted way of expressing the ELISA results in the case of experimental vaccines, since there is usually no uniform standard. Both end point and single dilution methods have significant disadvantages. In this paper, we obtained reproducible data with fewer dilutions of samples by addition of serially diluted standard serum to each ELISA plate. Since this ELISA method gives reliable antibody titer with less labor than other methods, it can strongly support vaccine development.

Effect of Eucalyptus Essential Oil on Respiratory Bacteria and Viruses

The activity of Eucalyptus globulus essential oil was determined for 120 isolates of Streptococcus pyogenes, 20 isolates of S. pneumoniae, 40 isolates of S. agalactiae, 20 isolates of Staphylococcus aureus, 40 isolates of Haemophilus influenzae, 30 isolates of H. parainfluenzae, 10 isolates of Klebsiella pneumoniae, 10 isolates of Stenotrophomonas maltophilia and two viruses, a strain of adenovirus and a strain of mumps virus, all obtained from clinical specimens of patients with respiratory tract infections. The cytotoxicity was evaluated on VERO cells by the MTT test. The antibacterial activity was evaluated by the Kirby Bauer paper method, minimum inhibitory concentration, and minimum bactericidal concentration. H. influenzae, parainfluenzae, and S. maltophilia were the most susceptible, followed by S. pneumoniae. The antiviral activity, assessed by means of virus yield experiments titered by the end-point dilution method for adenovirus, and by plaque reduction assay for mumps virus, disclosed only a mild activity on mumps virus.

Infectivity of Anticarsia gemmatalis nucleopolyhedrovirus to different insect cell lines: Morphology, viral production, and protein synthesis

The Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) is currently used as an efficient biological pesticide for the control of the velvetbean caterpillar (A. gemmatalis), an important pest of soybean in Brazil. Until now, production of the virus has been achieved mainly by infection of larvae on local soybean farms. Studies for the development of in vitro systems and the optimization of mass production in insects reared on artificial diets is now important to help to meet the actual demand for the bioinsecticide. We therefore, investigated the infectivity of AgMNPV in cell culture, which might contribute to the selection of suitable cell lines that may be used for in vitro production of this virus. The cytopathic effects induced by the virus, the production of viral particles and the synthesis of viral polypeptides were examined and compared in the cell lines from A. gemmatalis (UFL-AG-286), Trichoplusia ni (BTI-Tn-5B1-4 and TN-368), Spodoptera frugiperda (IPLB-SF-21AE and Sf9), Lymantria dispar (IPLB-LD-652Y), and Bombyx mori (BM-5). Whereas, Tn-5B1-4 and AG-286 cells produced large numbers of occlusion bodies, no polyhedra were visualized in either Ld-652Y or BM-5 cells, although extensive cell lysis was observed in BM-5. Analysis of the kinetics of viral protein synthesis by SDS–PAGE after pulse labeling with [35S]methionine, showed similar protein patterns in most of the cell lines tested. Exceptions were the LD-652Y and BM-5 cells, in which viral polypeptides, including polyhedrin, were not synthesized. In parallel, measurement of viral titers (budded virus) by the endpoint dilution method showed that Tn-5B1-4, AG-286, and SF-21AE cells were highly productive. Their TCID50 values, at 48 h p.i., were about 107 IU/ml. In addition to the lower formation of polyhedra, the viral titers determined in Sf9 and TN-368 cells were about 5 to10-fold lower. As expected, the viral titers obtained in LD-652Y and BM-5 cells were similar to basal levels.

Using QPCR to assign infectious potencies to adenovirus based vaccines and vectors for gene therapy: toward a universal method for the facile quantitation of virus and vector potency

The assignment of infectious potency to test articles of adenovirus has been conducted mainly using classical end-point dilution methods, which rely on virus induced cytopathology to reveal the presence of infectious virus. These assays suffer the disadvantages of labor intensity, duration, throughput restriction and variability. In the course of our development of an Ad5 based HIV vaccine for clinical evaluation, we sought a facile method for the assignment of potency to the numerous test articles generated during the development of bioprocesses for bulk manufacture, downstream purification and formulation. In this paper we describe a quantitative PCR based potency assay (QPA) which uses QPCR to quantitate adenovirus genomes replicated 24 h after the inoculation of a test article on 293 cell monolayers, and then relates that mass to potency by interpolation to a standard curve of replicated adenovirus genomes constructed with a reference adenovirus standard to which infectious potency has been previously assigned in the classical end-point dilution assay. The QPA assay for adenovirus is simple and rapid, with a throughput capacity adequate to the potency assay demands of bioprocess development, and with a precision expressed as a root variability of 16.8% R.S.D., allowing for close discriminations of the products of alternative process configurations. The adenovirus QPA principle can be applied to the quantitation of infectious potency of both RNA and DNA viruses and we report briefly on the development of QPA assays for measles and mumps. QPA assays owing to their simplicity and easy automation, rapidity, capacity and precision hold promise to become widely practiced methods for the quantitation of the potency of live virus vaccines and other recombinant virus vectors.

Improved growth of Flavobacterium psychrophilum using a new culture medium

The comparative study of different media used for cultivation of Flavobacterium psychrophilum showed that tryptone–yeast extract–salts supplemented with glucose, named FLP medium, was the most appropriate for the growth of this fish pathogen. By means of the endpoint dilution method, it was found that the bacterium grew more efficiently on the improved FLP medium than on the other four solid media assayed. This medium was also the most effective for the recovery of the pathogen from tissues of diseased fish. Culture of F. psychrophilum in FLP broth was also more rapid and provided high cell yields. In addition, FLP is a low-cost and easy-to-prepare medium.

Recognition of multiple Mycoplasma bovis antigens by monoclonal antibodies.

To produce monoclonal antibodies (MAbs), Balb/c AnN Crl BR mice were inoculated with the cell suspension of a Hungarian Mycoplasma bovis strain designated 26034. Three days after the last immunization the spleen of the immunized mouse was removed aseptically. The fusion of spleen cells with Sp2/0-Ag14 murine myeloma cells was performed in the presence of polyethylene glycol. The obtained hybrid cells were selected with hipoxantine, aminopterine and thymidine (HAT) medium. Two weeks after the fusion, the supernatants of the grown cells were tested by a self-developed indirect enzyme-linked immunosorbent assay (ELISA). The results showed that 63 antibody-producing hybridomas had been obtained. For accurate determination of the molecular weight of antigen determinants, the supernatants giving positive reaction in the ELISA were tested by Western blotting. According to the results, the obtained MAbs recognize the antigen determinants of the following molecular weights: 1B11: 63 kDa, 1C7: 63 kDa, 2C5: 22, 25 and 27 kDa, 2C9: 69 kDa, 3G12: 67, 69 and 72 kDa, 4H9: 63 kDa, 5B8: 22, 25 and 27 kDa, 5D3: 22, 25 and 27 kDa, 5C11: 69 kDa, 5E5: 22, 25 and 27 kDa, 6F11: 63 kDa, and 6H10: 22, 25 and 27 kDa. The 12 cell groups selected on the basis of the Western blotting were cloned twice by end-point dilution method. The cloned cells were propagated, and with 5 cell lines antibodies were produced in the CELLine bioreactor (Integra Biosciences, Zurich, Switzerland). Cell line 3G12 showed the highest productivity with an average daily output of 1.5 mg immunoglobulin. Cell line 5E5 produced 1.1 mg, 6H10 0.8 mg, 2C9 0.47 mg and 6F11 0.4 mg antibody per day. The isotype of the antibodies was determined by ELISA. The antibodies produced by the 12 cell lines tested were assigned to the IgG(1) subclass according to the heavy chain. Ten cell lines produced kappa and two produced lambda light-chain antibody. Possible cross-reactions of the produced monoclonal anti-M. bovis antibodies with certain Mycoplasma, Ureaplasma and Acholeplasma species were tested by an indirect ELISA procedure. All of the 12 antibodies tested gave a reaction with the antigen of M. bovis strain designated 26034. MAbs 3G12 (67, 69, and 72 kDa) and 5B8 (22, 25, and 27 kDa) gave no cross-reaction with antigens other than strains of the homologous Mycoplasma species. The other antibodies reacted with the M. bovigenitalium F7, M. anseris 8389, M. oculi, and M. gallisepticum s6 Holland antigens. Owing to its high specificity and affinity, the antibody produced by the cell line 3G12 is primarily considered suitable for use in immunodiagnostic tests of M. bovis infections.

Establishment of the first World Health Organization International Standard for human parvovirus B19 DNA nucleic acid amplification techniques.

A collaborative study, involving 26 laboratories from 14 countries, was carried out in order to establish a World Health Organization (WHO) International Standard for human parvovirus B19 (B19) DNA nucleic acid amplification techniques (NAT). Four samples: AA, BB (which were lyophilized), CC and DD (which were liquid preparations) were analysed using several different NAT assays. The mean B19 DNA content of each sample was determined for each laboratory using an end-point dilution method. There was good agreement between the overall mean 'equivalents'/ml obtained by the different assays. The mean log(10) 'equivalents'/ml were 5.76 for sample AA, 5.73 for sample BB, 5.82 for sample CC and 7.70 for sample DD. The differences in titre among samples AA, BB and CC were not statistically significant, but the titre of DD was significantly higher. Despite the range of NAT assays used in the study, it was possible to calculate the mean B19 DNA concentrations in the four preparations. Lyophilized preparation AA was established as the first International Standard for B19 DNA NAT assays and was assigned a concentration of 10(6) international units (IU)/ml.

The significance of hepatitis B virus DNA detected in hepatocellular carcinoma of patients with hepatitis C

Recently, it has been reported that hepatitis B virus (HBV) DNA is detected in cancerous liver tissues in some hepatitis B surface antigen negative chronic hepatitis C patients with hepatocellular carcinoma (HCC). However, the significance of HBV DNA detected in such cases remains unclear. The authors determined the presence or absence and the titers of HBV DNA in the liver tissue of anti-hepatitis C virus (HCV) positive/HBs antigen negative patients with HCC by polymerase chain reaction (PCR) and Southern hybridization, and correlated them with clinicopathologic parameters. HBV DNA was found in cancerous liver tissues from 12 (50.0%) of the 24 patients studied. However, Southern hybridization of genomic DNA in the cancerous liver tissues led to detection of HBV DNA in only 3 (12.5%) of the 24 patients, suggesting that the copy number of HBV DNA may be very low in most cases. Indeed, the titration of HBV DNA in cancerous liver tissues performed by the end point dilution method revealed that most contained less than one copy of HBV DNA per cell, although cells of monoclonal origin carrying integrated HBV DNA should have at least one copy of it. There was no significant difference in HBV DNA positivity between the HCC patients with and without underlying cirrhosis. The results of this study corroborate previous reports of frequent detection of HBV DNA in the liver tissue of anti-HCV positive/HBs antigen negative patients with HCC, but do not support an essential role of HBV in hepatocarcinogenesis in patients with chronic hepatitis C and occult HBV infection.