Phagocytosis and intracellular killing of the contagious equine metritis organism by equine neutrophils in serum.

Equine neutrophils were combined with Haemophilus equigenitalis (contagious equine metritis organism; CEMO) or Escherichia coli in low- and high-antibody-titer serum to evaluate the neutrophils ability to phagocytize and kill these bacteria. More E. coli than CEMO were phagocytized at each time period. After 120 min in low-antibody-titer serum, 56.3% of the E. coli and 34.3% of the CEMO were phagocytized. A total of 45% of CEMO and 74.9% of E. coli were phagocytized by 120 min when neutrophils were in high-antibody-titer serum. More than 75% of the ingested E. coli and 90% of the ingested CEMO were killed within 210 min of incubation. Fewer E. coli than CEMO were killed at any given time period. Ultrastructural examination showed CEMO to be degraded in the neutrophil. Degradation was the most extensive in neutrophils in high-titer serum. It is suggested that CEMO is a pathogenic extracellular bacterium incapable of prolonged intracellular survival and that it is slower to be phagocytized than a nonpathogenic E. coli.     

Determination of serum thyroxine using a resin sponge technique

A method is described for the determination of serum thyroxine using ion-exchange resin sponges. The principle is based on the use of protein-binding sites and part of the technique is similar to the Triomet resin uptake test. An isotope extraction recovery procedure is incorporated in the method. Studies of factors influencing the analysis are reported and the results of tests of accuracy, precision, and specificity are presented. Serum thyroxine iodine values are compared with protein-bound iodine estimations. The values found in health and disease are similar to the results of previous workers.The specificity of the analysis represents a considerable advantage over protein-bound iodine determinations since no interference is caused by iodides, iodine-containing contrast media, mercurial diuretics, mono-iodotyrosine, di-iodotyrosine, di-iodothyronine, and also tri-iodothyronine in physiological amounts. Therapy with diphenylhydantoin and tri-iodothyronine will tend to depress protein-bound iodine levels and thyroxine values are similarly influenced.     

Development of a poliovirus neutralization test with poliovirus pseudovirus for measurement of neutralizing antibody titer in human serum

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV.     

Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac. The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.     

Radioimmunoelectrophoretic identification of poliovirus inhibitors and their characteristic mode of action

Summary Poliovirus inhibitors present in normal equine and bovine sera were studied by means of radioimmunoelectrophoresis and column chromatography. The inhibitors in individual equine sera were identified as either one of the three immunoglobulins, M, A(T) and G, or mixtures of them, while those in bovine sera were found to be either one of M and G globulins, or the mixture of the two.The inhibitors belonging to different classes of immunoglobulin differed in their mode of action against the virus. M inhibitors were characterized by distinct activities in the kinetic neutralization and the plaque reduction tests. In contrast, A(T) inhibitors were highly active in the plaque-reduction and the precipitation-in-agar-gel tests, although they were inactive in the kinetics of neutralization. There were two types of G inhibitors. One did not neutralize the virus in the kinetic test, despite its distinct virus-precipitating activity. The other could neutralize but not precipitate the virusin vitro.Avidity of the inhibitors as revealed by dissociation of virus-inhibitor complexes at acidic pH's also differed among the immunoglobulin classes of the inhibitors. The complex formed with the NaIO₄-treated A(T) inhibitor was most stable and was not dissociated even at pH as low as 2.0.This study was supported in part by a grant for the Fundamental Scientific Research from the Education Ministry of Japan.The outline of this article was presented at the First International Congress for Virology, Helsinki, 1968.     

A simple purification and fluorescent assay method of the poliovirus 3C protease searching for specific inhibitors

Picornaviruses such as poliovirus, foot-and-mouth disease virus, and encephalomyocarditis virus produce their proteins by translating their genomic RNA, injected within the host cell, into a precursor polyprotein, which is then subjected to precise processing. The polyprotein is cleaved into mature proteins predominantly by the viral 3C protease. A simple purification and assay method for poliovirus 3C protease for use for screening for inhibitors of the 3C protease is described. A poliovirus cDNA fragment containing the 3C protease coding region was inserted into pET22b vector and expressed in Escherichia coli. The His-tagged protein (3CD'-His) was purified by a Ni-affinity column and the activity of the purified enzyme was measured by a fluorescent assay with a fluorogenic substrate containing the 3C-specific cleavage site, MocAc-MEALFQGPLQY-Dnp. The kinetic parameters calculated from the Lineweaver-Burk plot and the effects of inhibitors showed that E. coli expression with His tag and the assay using the fluorogenic substrate are efficient, simple and sensitive methods for purifying the 3C protease, and measuring its activity.     

Study of the cross-reaction between rabbit anti-bovine serum albumin antibodies and equine serum albumin

Cross-reactions between bovine serum albumin and equine serum albumin were studied using heterologous soluble complexes and specifically purified cross-reacting antibody.

Experiments with soluble complexes showed that homologous antigen can displace heterologous antigen specifically bound to antibody but heterologous antigen cannot displace homologous antigen. On gel precipitation tests a specific precipitation resulted when heterologous soluble complex reacted with homologous antigen.

By using equine serum albumin conjugated to polyaminopolystyrene the cross-reacting antibodies from anti-bovine serum albumin imune sera could be isolated. These are divalent 7S, γ-globulin antibodies. A figure of cross-reaction was obtained when these purified antibodies were tested by double diffusion in agar with bovine and equine serum albumins.

The results obtained both with soluble complexes and with purified antibody support the view that cross-reacting antibody is more avid for the homologous than for the heterologous antigen.

     

A rapid method for quantitative assay of poliovirus from water with the aid of the fluorescent antibody technique

Summary A method is described for the rapid quantitative demonstration of polioviruses in water with the aid of the fluorescent antibody technique. Identification of the virus is possible after 18–24 hours as compared to 3–5 days required with the plaque count method. Approximately 10 plaque forming units, concentrated from a volume of 40 liters of seeded tap water could be demonstrated by the rapid method. Positive cells were already seen after 6–9 hours; the results were, however, not sufficiently quantitative. The method also showed itself to be less susceptible to bacterial contamination than the current isolation methods. Its possible utilization as a rapid, primary test for viral contamination of potable water is discussed.With 1 Figure     

Automatic technique for titration of influenza virus haemagglutination inhibitors

An automatic method, utilizing the AutoAnalyzer, for titration of influenza virus haemagglutination inhibitors is described. The titres obtained compare favourably with those obtained by densitometric methods. So far, the technique has not proved superior to the densitometric methods in either speed of operation or in reproducibility but various developments are suggested to effect this.