Presence Of Antiserum Research Articles

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Graphene oxide arms oncolytic measles virus for improved effectiveness of cancer therapy

BackgroundReplication-competent oncolytic viruses (OVs) have been proven to be a potent anticancer weapon for clinical therapy. The preexisting neutralizing antibody in patients is a big challenge for oncolytic efficacy of OVs. Graphene oxide sheets (GOS) possess excellent biological compatibility and are easy to decorate for targeted delivery.MethodsWe generated PEI-GOS-PEG-FA (Polyethyleneimine-Graphene oxide sheets-Polyethylene glycol-Folic acid). After intravenous injection, the distribution of PEI-GOS-PEG-FA in tumor-bearing mice was visualized by the IVIS Lumina XR system. Then, the oncolytic measles virus (MV-Edm) was coated with PEI-GOS-PEG-FA to form a viral-GOS complex (GOS/MV-Edm). The oncolytic effects of GOS/MV-Edm were investigated both in vitro and in vivo.ResultsGOS/MV-Edm exhibited higher infectivity and enhanced oncolysis. In tumor-bearing mice, GOS/MV-Edm had significantly elevated viral replication within the tumor mass, and achieved an improved antitumor effect. Then, we confirmed that GOS/MV-Edm entered cancer cells via the folate receptor instead of CD46, a natural cognate receptor of MV-Edm. GOS/MV-Edm remained the infectivity in murine cells that lack CD46. Finally, we found that GOS/MV-Edm was effectively protected from neutralization in the presence of antiserum both in vitro and in vivo. In passively antiserum immunized tumor-bearing mice, the survival was remarkably improved with intravenous injection of GOS/MV-Edm.ConclusionOur findings demonstrate that GOS/MV-Edm displays significantly elevated viral replication within the tumor mass, leading to an improved antitumor effect in solid tumor mouse model. Our study provided a novel strategy to arm OVs for more efficient cancer therapy. That may become a promising therapeutic strategy for cancer patients.

Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay

Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

Enhanced infectivity of H5N1 highly pathogenic avian influenza (HPAI) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage

Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-γ. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-γ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-γ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin.

Immunological and biological relationship among flagellin of Pseudomonas aeruginosa, Burkholderia cepacia, and Stenotrophomonas maltophilia

The flagellar protein (flagellin) was isolated and purified from strains of Pseudomonas aeruginosa, Burkholderia cepacia and Stenotrophomonas maltophilia. A significant difference was observed in the molecular weight of different flagellin preparations obtained from these bacterial isolates. Antiserum prepared against S. maltophilia flagellin did not react with flagellin of P. aeruginosa or/and B. cepacia on Immunoblot or in indirect ELISA. In addition the anti-flagellin did not agglutinate P. aeruginosa and B. cepacia. No inhibition of motility of P. aeruginosa and B. cepacia was observed in presence of antiserum; though the latter inhibited the motility of S. maltophilia. The results of the present study prove that no specific relationship existed among all the studied flagellar proteins obtained from closely related bacteria.

ATP-dependent fructose uptake system in Deinococcus radiodurans

The bacterial phosphoenolpyruvate (PEP)-dependent group translocation system (PTS) requires the presence of both membrane-bound and cytoplasmic components to phosphorylate and translocate sugar. Deinococcus radiodurans has a functional fruA gene coding for the membrane-bound components of the fructose-specific PTS. However, fruB gene coding for the fructose-specific cytosolic components of PTS is a pseudogene. Yet, this bacterium metabolized fructose readily. In vitro studies showed that both cell membranes and cytoplasmic fractions of the cells were needed for fructose phosphorylation. Further studies showed that fructose phosphorylation required ATP, not PEP, as the phosphate donor. Unlike most PEP-dependent PTS systems, fructose phosphorylation is sensitive to sodium fluoride, a kinase inhibitor. Fructose phosphorylation was also inhibited in the presence of antiserum against a kinase phosphorylation site. Rhodobacter capsulatus has a functional fruA-fruB system. Complementation assays by reconstituting the membrane fraction of D. radiodurans to the cytoplasmic fraction of R. capsulatus resulted in a PEP-dependent fructose phosphorylation, whereas mixing the membranes of R. capsulatus and the deinococcal cytosol in vitro resulted in an ATP-dependent fructose phosphorylation.

Characterization of fertility related antisperm antibodies— a step towards causal treatment of immunological infertility and immuno-contraception

Antisperm antibodies (ASA) occur in men and women and may significantly impair fertility. The identification of functionally relevant antigens of ASA is important for establishing causal treatment options on the one hand and may, on the other hand, enhance the development of tools for immunocontraception. One candidate protein is addressed in an article of Wang et al. 1 published in Asian Journal of Andrology. The authors report results of animal experiments where that antibodies against nuclear autoantigenic sperm protein (NASP), a histone-binding protein, were investigated for their antifertility effect. Mice were immunized with a recombinant mouse NASP and a synthetic human NASP. It has been demonstrated that immunization with both NASP-variants can inhibit sperm-egg binding as well as in vitro fertilization (IVF). Moreover, this antibody interferes with the fertility of immunized animals as shown by a reduced litter size. The testis is an immunologically privileged site where germ cell antigens are protected from autoimmune attack 2, 3. However, due to disruption of the blood-testis barrier occurring from testicular injury, or as a consequence of trauma to the epididymis or vas deferens many testicular isoenzymes and other proteins get auto-antigenic during immunological challenges resulting in the formation of ASA in the blood serum, seminal plasma or located on the sperm membrane. ASA have also been reported to be associated with inflammation, cryptorchidism, varicocele and surgical intervention in the genital organs 4. ASA may interfere with different sperm functions essential for the fertilization process. However, the problem is to detect whether or not ASA of an individual man are of functional relevance, i.e. whether the ASA binding to a sperm surface antigen also influences sperm function and which one. In previous studies several antibody-binding proteins have been characterized in isolated sperm surface membranes and several proteins have been described whose ASA could be associated with agglutinations 5, motility 6, cervix mucus penetration 7, acrosome reaction 8, zona binding 9 and penetration and oolemma binding 10. NASP has been found to be important for the pronucleus formation to which most vasectomized men develop autoantibodies 11. In the present study several important results regarding NASP have been obtained: 1. Sperm-egg binding and IVF of mouse oocytes with capacitated mouse spermatozoa were inhibited in the presence of antisera against NASP. 2. Anti-NASP did not change sperm motility. 3. Sera from infertile men containing antisperm antibodies reacted with anti-NASP. 4. After immunization of fertile female mice with NASP the pregnancy rate was significantly reduced and the reduction in litter size correlated with the antibody-titer. Depending on the immunogen used (recombinant mouse NASP) or a synthetic peptide (human NASP), the immunized mice regained fertility at rates comparable to those of the control mice after the antibody titer had declined or the animals remained infertile (immunized with mouse NASP). With regard to the mechanism by which fertility is disturbed—inhibition of sperm-egg binding and IVF—it is speculated that the antibody effect must be associated with the cell surface and not with the cytoplasm or nucleus. This is a little bit in contrast to the fact that NASP has been shown to be located mainly over the nucleus and only partly in the acrosome 12: here the authors discuss that some part of the NASP may be exposed for a short time when it is carried into the ovum with the sperm nucleus at fertilization. This question can be answered after performing intra cytoplasmic sperm injection (ICSI), which will be the next step in a further study announced by the authors in order to investigate the direct effects of the anti-NASP antibodies on embryo development and implantation. As ICSI is suggested as the appropriate treatment to overcome antisperm antibodies'action on the one hand, and—according to my own experience—fertilization and pregnancy rates are lower in couples with male immunological infertility compared to couples without antisperm antibodies in the male, even when ICSI is applied, it will be very interesting to see what these future studies will reveal. They will probably show that it is not an antibody against a single sperm antigen that causes a significant reduction of fertility, but is more likely multiple ASAs that contribute to an individual fertility problem. Future results will increase our understanding of the specific mechanisms that elicit the autoimmune response and of the active profile of ASA that leads to an antibody-mediated infertility. Thus, specific therapies based on the use of monoclonal antibodies as well as new approaches to male immune contraception may be developed.

Immunochemical investigation of human seminal plasma acid phosphatase.

Acid phosphatase of human seminal plasma is inhibited in presence of normal rabbit serum involving non-competitive mechanism whereas this character is normalized and activity is stabilized in presence of its rabbit antiserum. Kinetic studies on stabilization of acid phosphatase by antiserum were combined by L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate, a specific inhibitor of prostatic acid phosphatase. L-tartrate inhibited the enzyme in presence and absence of normal and antiserum by competitive mechanism and percentage of inhibition of L-tartrate was involve in the binding with acid phosphatase antibodies. The kinetics of heat denaturation of acid phosphatase suggested that seminal acid phosphatase is more heat labile in presence and absence of normal serum than in presence of antiserum. The temperature of denaturation corresponding to half the initial activity(T 1/2) in presence of normal and antisera were 34 degrees and 66 degrees C respectively. NOn-linearity of Arrhenius plots for denaturation rates of the enzyme suggested various conformational states for denaturation yielding Ea varying between 20.08 and 5.03 Kcals at 25-65 degrees C.

Dodecamer is Required for Agglutination of Litopenaeus vannamei Hemocyanin with Bacterial Cells and Red Blood Cells

Hemocyanins are multi-functional proteins, although they are well known to be respiratory proteins of invertebrate to date. In the present study, the agglutination ability of two oligomers of hemocyanin, hexamer and dodecamer, with pathogenic bacteria and red blood cells (RBCs) is investigated in pacific white shrimp, Litopenaeus vannamei. Hexameric hemocyanin exhibits an extremely high stability even in the absence of Ca(2+) and in alkaline pH. Dodecamer (di-hexamer) is easily dissociated into hexamers in unphysiological conditions. Hexamer and dodecamer are interchanged reciprocally with environmental conditions. Both oligomers can bind to bacteria and RBCs, but agglutination is observed only using dodecamer but not using hexamer in agglutination assay. However, the agglutination is detected when hexamer is utilized in the presence of antiserum against hemocyanin. These results indicate that dodecamer of hemocyanin is required for agglutination with bacteria and RBCs. It can be logically inferred that there is only one carbohydrate-binding site to bacterial cells and RBCs in the hexamer, while at least two sites in the dodecamer. Our finding has provided new insights into structural-functional relationship of hemocyanin.

Adaptability costs in immune escape variants of vesicular stomatitis virus

We have used vesicular stomatitis virus (VSV) to determine the cost of antiserum resistance during escape from a polyclonal immune response. Replication of VSV in the presence of polyclonal antiserum resulted in the selection of antibody-escape mutants, as shown by increased fitness in the presence of antiserum and by increased resistance to neutralization. However, resistance came at a cost of overall fitness loss in the BHK-21 host cells. Sequencing of the surface G glycoprotein showed that two to four mutations were fixed in each population, most of which mapped in the A1 and A2 antigenic sites. Selected resistant populations were passaged as large populations in BHK-21 cells under constant conditions, which would normally lead to fitness increases. Nevertheless, many of the populations showed little or no sign of recovery, although the resistant phenotype was maintained. These results suggest that while antiserum resistance can develop, it may come at a cost in fitness and further limitations in the adaptability of the populations.

Immunocytochemical localization of antigens recognized by human antisera in infective larvae of Wuchereria bancrofti.

Ultrathin sections of L3 of Wuchereria bancrofti embedded in hydrophilic resin were incubated with antisera pools from individuals (1) asymptomatic microfilaremic with different microfilaria (mf) densities (1-100, 101-500, and >1,000 mf/ml); (2) chronic with hydrocele or lymphedema; and (3) with no evidence of microfilaremia or clinical filariasis but residing in an endemic area. The groups of microfilaremic subjects studied presented differences relative to the intensity of labeling, with the density of gold particles per square micrometer proportional to microfilaremia. Incubation of ultrathin sections of W. bancrofti L3 larvae in the presence of antisera from patients exhibiting chronic obstructive lymphatic pathology of hydrocele and from individuals with clear clinical evidence of lymphedema exhibited a strong reaction in the same tissues. Except for the endemic normal group, all groups studied showed reactivity against epitopes in all tissues of infective larvae of W. bancrofti. The cuticle presented an intense labeling, suggesting a possible target structure for immune response.

AN INFLUENCE OF STRESS PROTEIN CSP 310 AND ANTISERUM AGAINST THIS PROTEIN ON LIPID PEROXIDATION IN CEREAL MITOCHONDRIA

It is determined that an addition of an anti-CSP 310 antiserum to isolated winter wheat and maize mitochondria caused more significant increasing of spontaneous lipid peroxidation than the addition of stress protein CSP 310. It is shown that, at function of different mitochondrial respiratory chain complexes, the lipid peroxidation in winter wheat and maize mitochondria take place with different intensities. Under the functioning of mitochondrial respiratory chain complex IV, the maximum output of lipid peroxidation products, dienic conjugates is detected. The presence of antiserum against CSP 310 in incubation media induces lipid peroxidation more than the presence of CSP 310 in mitochondria isolated from stressed plants under these conditions. Based on data obtained, it is possible to conclude that in vivo endogenous CSP 310, during a cold stress, has an antioxidant activity the same as other known uncoupling proteins.

Growth promoting effects of human placental lactogen during early organogenesis: a link to insulin-like growth factors.

Many maternally derived factors may be involved in the regulation of embryonic growth but the control mechanisms involved are poorly understood. Human placental lactogen (hPL) has been implicated in playing a role in the control of embryonic growth. Several investigators suggested that there may be a possible link between the effects of this hormone and insulin-like growth factors (IGFs). In order to determine the growth promoting potential of hPL and involvement of IGFs in the mechanism of action of the hormone, 9.5 d rat embryos were cultured in vitro for 48 h in depleted serum in the presence and absence of hPL with additional IGF antisera. The growth supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. Addition of hPL (3.2-25.6 ng/ml) to depleted serum significantly improved embryonic growth and development, suggesting that the developing embryo may utilise hPL. The presence of antisera against hPL, IGF-I and -II abolished the hPL-induced increase in the development in all parameters suggesting that there may be a possible link between the IGFs and the effects of hPL on rat embryonic development and this hormone may achieve its growth promoting effects via IGFs.

Properties of Streptococcus sanguinis Glucans Formed Under Various Conditions

The aim of our study was to determine whether the structure of glucans formed by glucosyltransferase from Streptococcus sanguinis (GtfSs) on a surface differ from those formed in solution and to explore the effects of antiserum to Gtfs, control normal rabbit serum, starch hydrolysates (STH) and dextran on S. sanguinis (GtfSs) glucan. Linkage analyses showed that solution–formed glucans are predominantly α–1,6–linked and have a small amount of α–1,3–linked glucose. Surface–formed glucans have enhanced susceptibility to mutanase. Solution– and surface–formed glucans made in the presence or absence of sera, STH, and dextran contain linkages which differ in both amount and type from control glucans. The GtfSs enzyme in solution exposed to antiserum behaves as if it is adsorbed to a surface. Binding of Streptococcus mutans GS–5 and Actinomyces viscosus OMZ105E (Ny1) to S. sanguinis glucan differs if the glucan is formed in the presence of antiserum. The information could help to define the role of glucans in the formation of pellicle, colonization of tooth surfaces and the accumulation of dental plaque.

Characterization and Purification of Carbohydrate Response Element-Binding Protein of the Rat L-Type Pyruvate Kinase Gene Promoter

The L-III transcriptional regulatory element of the rat pyruvate kinase L gene is located between −170 and −150 base pairs upstream of the hepatocyte-specific transcription initiation site. As the L-III element is not only necessary for cell type-specific expression but also for transcriptional stimulation by carbohydrates, it is also referred to as a carbohydrate-response element. Electrophoretic mobility shift assays using rat liver nuclear extract showed that L-III element-binding protein (L-IIIBP) was observed as multiple bands. These bands disappeared when the nuclear extract was preincubated at 60°C for 5 min and were competed with unlabeled L-III oligonucleotide but not with unlabeled adenovirus major late promoter E box oligonucleotide. In addition, these bands were not affected in the presence of antiserum against upstream stimulating factor (USF). Thus, we conclude that L-IIIBP is different from USF. Then, heat-labile L-IIIBP was purified from rat liver nuclear extracts. Purified L-IIIBP exhibited two bands on sodium dodecyl sulfate/polyacrylamide gel electrophoresis by silver staining. Ultraviolet crosslinking experiment showed that both bands had binding activity to the L-III oligonucleotide.

Adjuvant effects and antiserum action potentiation by a (herbal) compound 2-hydroxy-4-methoxy benzoic acid isolated from the root extract of the Indian medicinal plant `sarsaparilla' ( Hemidesmus indicus R. Br.)

The adjuvant effect and antiserum potentiation of a compound 2-hydroxy-4-methoxy benzoic acid were explored in the present investigation. This compound, isolated and purified from the Indian medicinal plant Hemidesmus indicus R. Br, possessed antisnake venom activity. Rabbits immunized with Vipera russellii venom in the presence and absence of the compound along with Freund's complete adjuvant, produced a precipitating band in immunogel diffusion and immunogel electrophoresis. The venom neutralizing capacity of this antiserum showed positive adjuvant effects as evident by the higher neutralization capacity (lethal and hemorrhage) when compared with the antiserum raised with venom alone. The pure compound potentiated the lethal action neutralization of venom by commercial equine polyvalent snake venom antiserum in experimental models. These observations raised the possibility of the use of chemical antagonists (from herbs) against snake bite, which may provide a better protection in presence of antiserum, especially in the rural parts of India.

Attachment ofStreptococcus gordoniiHG 222 toStreptococcus oralisNy 586 and the Influence of Saliva

The binding of Streptococcus gordonii HG 222 to other streptococci was tested by coaggregation and by binding to immobilised bacteria in an ELISA assay. Out of 17 streptococcal strains, HG 222 coaggregated only with Streptococcus sanguis HG 1470 and Streptococcus oralis Ny 586. Binding of HG 222 to immobilised bacteria also occurred only with HG 1470 and NY 586. Binding of HG 222 to S. oralis Ny 586 was further investigated. Preincubation of Ny 586 coated microtitreplates with whole saliva, parotid, submandibular and sublingual saliva enhanced binding of HG 222 to Ny 586. Several salivary substances bound to both HG 222 and Ny 586, namely low molecular weight mucins, salivary agglutinin, and IgA. There were indications that binding of HG 222 to Ny 586 was especially enhanced by IgA. Binding of IgA to both bacteria could be demonstrated with all the types of glandular saliva. In addition, purified colostral IgA enhanced binding of HG 222 to Ny 586. Saliva preincubation of Ny 586 coated microtitreplates in the presence of antiserum against the IgA a-chain inhibited saliva mediated binding of HG 222 to Ny 586. In this case, binding of salivary agglutinin to Ny 586 was also inhibited, suggesting complexation between IgA and agglutinin. In conclusion, these results indicate that colonisation of dental surfaces by S. gordonii may be accomplished by binding to strains of S. oralis or S. sanguis . This process may be enhanced by IgA in saliva. Keywords: oral streptococci; saliva; coaggregation; interbacterial binding.

Effects of Typhus Rickettsiae on Peritoneal and Alveolar Macrophages: Rickettsiae Stimulate Leukotriene and Prostaglandin Secretion

Monolayers of mouse resident peritoneal macrophages were incubated with typhus rickettsiae, and macrophage secretion of leukotriene B4 (LTB4) was assessed. Macrophages incubated with native rickettsiae, but not those incubated with hemolytically inactivated rickettsiae, secreted significantly more LTB4 than did sham-treated macrophages. Antirickettsial antiserum was opsonic and blocked hemolysis, and macrophages incubated with rickettsiae in the presence of antiserum did not secrete more LTB4 than those incubated with buffer alone. Native rickettsiae also stimulated alveolar macrophages to secrete LTB4 and prostaglandin E2 (PGE2), but inactivated rickettsiae had no effect. Finally, because trifluoperazine did not alter macrophage LTB4 secretion in the presence of rickettsiae, but inhibited PGE2 secretion, it was suggested that the mechanisms by which rickettsiae stimulated production of these autacoids may have differed.

Detection of Immunoprecipitated Systemic Lupus Erythematosus Antigens by Immunoblot Analysis

When antigens are isolated from staphylococcal protein A immunoprecipitation pellets for analysis by SDS polyacrylamide gel electrophoresis and immunoblotting, severe background problems, due to the presence of antisera and bacterial proteins, can result. We describe a procedure for the analysis of immunoprecipitated systemic lupus erythematosus antigens (e.g., La, Ro, and Sm) which significantly reduces this background while retaining sensitivity with respect to antigen detection. We have adapted a method previously described (MacSween, J.M. and Eastwood, S.L. Methods Enzymol. 1981; 73:459-471) in which lithium diiodosalicylate is used to separate the immunoprecipitated antigen from a covalent antibody-staphylococcal protein A complex. In addition, a modified series of immunoblot incubations was employed, in which antigenic proteins were identified by incubating blots with the antiserum used for the original immunoprecipitation (e.g., La) followed by protein A-biotin and avidin-alkaline phosphatase. Overall, the procedure is straight-forward and may be applicable to other immunoblot systems.

Persistent Infection of MDCK Cells by Influenza C Virus: Initiation and Characterization

Persistent influenza C virus infection was readily initiated in Madin-Darby canine kidney (MDCK) cells at low m.o.i. and has been maintained for over 1 year. The persistently infected (p.i.) cultures were characterized by the following properties: virus infection was limited to a minority of cells, small amounts of infectious virus were produced together with low levels of interferon (IFN) and the cultures were resistant to superinfection by homologous virus and vesicular stomatitis virus, but not by influenza A and B viruses. These properties fluctuated cyclically with passage of the p.i. culture. When p.i. cultures were cured by cultivation in the presence of antiserum, the cultures lost their IFN-producing activity and became as susceptible to homologous virus as normal MDCK cell culture. The results suggest that persistent influenza C virus infection may be regulated by endogenously produced IFN. Under the condition of high m.o.i. a persistent influenza C virus infection could not be initiated in MDCK cells due to the development of cytopathic effects.