Inhibition of subtilisin by influenza virus infected allantoic fluids.

Allantoic fluids harvested from embryonated chicken eggs infected with reference strains of influenza A viruses were analysed for subtilisin inhibitor activity. While all acid heat-treated and nontreated virus-infected fluids could reduce subtilisin activity, fluids of FM and Bangkok strains had the greatest inhibitory ability. The degree of subtilisin inhibition closely paralleled the appearance of infectious Bangkok and FM virus in allantoic fluid. Maximum levels were achieved at 48 hr post-infection (p.i.) Ultracentrifugation analyses indicated that the bulk of thermostable inhibitor(s) of 48 hr Bangkok and FM infectious fluids remained in the supernatant rather than sedimenting with the viral pellet.

Allantoic fluid protease activity during influenza virus infection.

Neutral protease activity of allantoic fluid from embryonated chicken eggs was quantified during the course of influenza virus infection. Antigenic subtypes of influenza A viruses selected for study were H1N1 strains PR/8/34, Brazil/8/78, FM/1/47, the H3N2 strain Bangkok/1/80 and the H5N9 Turkey/ /Ontario/66 as well as the Sendai strain of parainfluenza type 1 virus. Three different types of profiles of allantoic fluid proteases could be readily distinguished after infection of eggs with various virus strains. In all profiles, periodic peaks of protease activity always preceded the partial shut down of protamine cleaving proteases which paralleled the production of near maximum titers of infectious virus. To determine the mechanism involved in this reduction of proteolytic activity, infectious allantoic fluids were analysed for the presence of protease inhibitors. Acid heat treated 48 hour virus-infected allantoic fluids of different influenza strains could inhibit the activities of subtilisin and allantoic fluid proteolytic enzymes.

Effects of influenza virus replication on neutral proteolytic activities in embryonated egg fluids.

The levels of neutral protease activity associated with allantoic and amniotic fluids of embryonated eggs during the replication of influenza strains A/PR/8/34 (H1N1) and A/turkey/Ontario/7732/66 (H5N9) were investigated. A sensitive fluorometric technique proved useful for characterization and monitoring changes of protease activities in egg fluids. The predominant type of protease in allantoic and amniotic fluids had trypsin-like specificities. Variation in protease levels of both fluids occurred throughout the course of virus replication irrespective of the virus strain or the route of inoculation used. Concomitant with the production of high levels of infectious virus there was a marked decrease in neutral protease activity in the fluid from the cavity initially infected. Translocation of virus also occurred especially with amniotically infected eggs, as evidenced by high infectious virus titers and decreased protease activities in allantoic fluids.

Protease susceptibility of human A influenza virus polypeptides.

The proteolytic susceptibility of polypeptides of four antigenically distinct subtypes of influenza a virus strains of human origin was studied. The extent of degradation of polypeptide molecules of strains A/PR/8/34 (H0N1) (PR), A/FM/1/47 (H1N1), A/Singapore/1/57 (H2N2) and A/Hong Kong/8/68 (H3N2), assessed by densitometry of gels after sodium dodecylsulfate polyacrylamide gel electrophoresis was variable by treatment with trypsin. Also, sequential treatment of PR strain initially with phospholipase D followed by proteases of different specificities suggested differences in susceptibility of surface and internal polypeptide molecules. The significance of these results is discussed.

Inhibition of vesicular stomatitis virus multiplication in Vero cells by 2-deoxy-D-glucose.

2-Deoxy-D-glucose (DOG) effectively inhibited vesicular stomatitis virus (VSV) multiplication in Vero cells when pyruvate-containing medium was used as an energy source. The effectiveness of the antimetabolite was markedly reduced by substituting glucose for pyruvate in the maintenance medium. Addition of DOG at intervals during the viral growth cycle caused a notable decrease in viral yields. This inhibiting effect was reversed by mannose and to a lesser extent by glucose. VSV-RNA synthesis was greatly reduced, thereby eventually resulting in decreased levels of virus proteins. Polyacrylamide gel electrophoresis of purified VSV grown in medium containing pyruvate and DOG revealed the presence of two peaks in the region of the virus G protein. Possibly, DOG induces the synthesis of aberrant viral proteins which become incorporated into the viral membrane, resulting in noninfectious particles.

Diminished virucidal activity of kethoxal against vesicular stomatitis virus pretreated with guanidinating reagent and proteases.

The reactivity of vesicular stomatitis virus (VSV) with kethoxal can be appreciably altered by treatment with 1-guanyl-3, 5-dimethyl pyrazole nitrate (GDMP) and proteolytic enzymes. Pretreatment of purified VSV with GDMP or proteolytic enzymes markedly reduced the effectiveness of kethoxal as a virucide. The rate of neutralizability of GDMP- and trypsin-treated viruses by specific antiserum differed from that of controls.

Adenosine triphosphate phosphoydrolase activity associated with purified parainfluenza type 3 virions.

An adenosine triphosphate phosphohydrolase associated with purified parainfluenza type 3 virions has been characterized. It hydrolyzed ATP to ADP and AMP when activated with Mg-2+ ions. Using Ca-2+ the production of ADP was inhibited but not that of AMP. Neither K+ NOR Na+ ions were required for the expression of maximal activity. Ouabain had no inhibitory effect on enzyme activity even at 10-3M. After exposure of virus preparations to Tween 20, enzyme activity was not affected. A linear relationship between enzyme activity and concentration of virus was observed.

Immunoglobulins and their relative neutralizing efficiency in cattle immunized with infectious bovine rhinotracheitis--parainfluenza-3 (IBR-PI-3) virus vaccine.

The localization of anti-IBR and anti-PI-3 activity in the serum and nasal secretory immunoglobulins following intranasal immunization of cattle with a mixed vaccine (IBR-PI-3, MLV, TCO) was studied and was found to reside in the nasal secretory IgA, serum IgM and IgG fractions. The computation of their relative virus neutralizing efficiencies from kinetic data revealed their order of neutralizing efficiencies to be IgM greater than IgA greather than IgG.

Inhibition of vesicular stomatitis virus replication by 9-beta-D-arabinofuranosyladenine.

9-beta-d-Arabinofuranosyladenine (Ara-A) effectively inhibited the production of infectious vesicular stomatitis virus (VSV) in MDBK cells. Furthermore, inhibition was shown to begin as early as the first 3 hr of infection. Studies employing (3)H-l-leucine indicated that Ara-A did not affect protein synthesis in uninfected cells, although it did cause a marked stimulation of protein synthesis in VSV-infected cells during the log phase of the growth cycle. Puromycin, an inhibitor of protein synthesis, was a more effective viral inhibitor than Ara-A. However, the combination of Ara-A and puromycin was less effective than puromycin alone except when present for long time periods. Short-term labeling experiments with (3)H-uridine demonstrated that Ara-A depressed ribonucleic acid (RNA) synthesis in uninfected cells, whereas periods of stimulation and depression of radioisotope incorporation occurred in infected cells. The results support the notion that Ara-A is incorporated into RNA early during viral replication.