Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.     

Proteolytic activity of bovine lactoferrin

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.     

C1q (c1) receptor on human platelets: inhibition of collagen-induced platelet aggregation by C1q (C1) molecules.

Hemolytically active human C1q incubated with EA before the addition of complement inhibited the immune hemolysis. On the contrary, heat-inactivated preparation (30 min 56 degrees C) was ineffective. Preincubation of EA with bovine collagen also resulted in a decreased hemolysis. When aggregation was measured by a turbidimetric method in citrated human platelet-rich plasma, it was found that hemolytically active human C1q (C1) alone does not induce platelet aggregation. However, in its presence the platelets failed to aggregate or exhibited a significantly reduced aggregation response to bovine collagen. The inhibition by C1q depended on the preincubation time with platelets. Heat treatment (30 min 56 degrees C) destroyed the inhibitory action of C1q (C1). The effect of C1q proved to be highly specific because different C1q preparations at their inhibitory doses in collagen-induced platelet aggregation did not influence the response to other aggregating agents (bovine thrombin, ADP, horse anti-human thymocyte globulin, goat anti-baboon platelet antiserum). The results prove that collagen and C1q are capable of binding to the same site(s); namely, to those of EA and human platelets; furthermore, they suggest the presence of a receptor for C1q (C1) on human platelets.     

Non-specific esterase activity in bovine acrosomes

Synopsis Fixed and unfixed smears of bovine spermatozoa were stained for esterase activity at pH 6.1–6.2 using 1-naphthyl acetate as substrate and hexazonium pararosaniline as coupler. Best results were obtained with unfixed smears prepared from suspensions of spermatozoa washed free of seminal plasma. The most intense staining was obtained when smears were incubated at 4°C for 40 hr with replacement of the incubating medium every 12 hr. No staining was detected in spermatozoa when smears were incubated in medium lacking substrate or when heat-inactivated smears were incubated in complete medium. Enzyme activity was restricted to the acrosomal cap; no final reaction product was detected in or on the post-nuclear cap, the mid-piece or the remainder of the flagellum. Enzyme activity was distributed quite uniformly throughout the acrosome with no indication of the existence of localized regions with high activity. The use of fixatives, especially those containing formalin, is not recommended for esterase studies of bovine spermatozoa.     

Effects of conditioned media from murine granulosa cell lines on the growth of isolated bovine preantral follicles

Isolated morphologically normal bovine preantral follicles (40 to 70 microm) were cultured for 8 d in collagen gel in control medium or in 1 of 3 conditioned media from the murine granulosa cell lines GRMO1L, GRMO2 and GE2. The percentages of follicles at Day 1 that remained nomal at Day 8 were similar for follicles cultured in the conditioned and control media (84 to 90%). A significantly higher percentage of follicles cultured in each of 3 conditioned media started to grow (89%; P < 0.05) and their increase in diameter was greater than that of follicles cultured in control medium (72%; P < 0.05). The mitotic activity of the granulosa cells from follicles cultured in conditioned media was increased (P < 0.05) indicated by a higher percentage of nuclei that incorporated BrdU compared with that of follicles cultured in control medium. Follicular viability was measured by the presence of nonspecific esterase activity, active mitochondria and dead cells in cultured follicles using the fluorescent probes calcein-AM, rhodamine 123 and ethidium homodimer-1 in combination with confocal laser scanning microscopy. The percentages of follicles with esterase activity and active mitochondria present in their granulosa were similar for follicles in all groups. Culturing in GRMO2 or GE2 tended to lower the number of granulosa with dead cells. The percentage of follicles with oocytes without esterase activity and active mitochondria was lower (P < 0.05) in follicles cultured in GRMO2 or GE2 compared with those cultured in control medium. Moreover, the percentages of dead oocytes tended to be higher in follicles cultured in GRMO1L and GE2 compared with oocytes of follicles incubated in control medium. Taken together, the conditioned media stimulated follicular growth and granulosa viability as well as enhance mitotic activity of the granulosa cells. However, they negatively affected oocyte viability.     

Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4

A novel hot spring thermophile, Anoxybacillus gonensis A4 (A. gonensis A4) was investigated in terms of capability of tributyrin degradation and characterization of its thermostable esterase activity by the hydrolysis of p-nitrophenyl butyrate (PNPB). It was observed that A. gonensis A4 has an esterase with a molecular weight of 62 kDa. The extracellular crude preparation was characterized in terms of substrate specificity, pH and temperature optima and stability, kinetic parameters and inhibition/activation behaviour towards some chemicals and metal ions. Tributyrin agar assay showed that A. gonensis A4 secreted an esterase and V(max) and K(m) values of its activity were found to be 800 U/L and 176.5 microM, respectively in the presence of PNPB substrate. The optimum temperature and pH, for A. gonensis A4 esterase was 60-80 degrees C and 5.5, respectively. Although the enzyme activity was not significantly changed by incubating crude extract solution at 30-70 degrees C for 1 h, the enzyme activity was fully lost at 80 degrees C for same incubation period. The pH-stability profile showed that original crude esterase activity increased nearly 2-fold at pH 6.0. The effect of some chemicals on crude esterase activity indicated that A. gonensis A4 produce an esterase having serine residue in active site and -SH groups were essential for its activity.     

The effect of monovalent cations on the pre-steady state reaction kinetics of bovine activated plasma protein C and des-1-41-light chain activated plasma protein C

A pre-steady state kinetic analysis of the stimulation by monovalent cations of the activity of bovine activated protein C (APC) and a proteolytic fragment of APC, des-1-41-light chain activated protein C (GDAPC), toward the substrate, 4-methylumbelliferyl p-guanidinobenzoate, has been undertaken. With the cations Na+ and Cs+, at least two cation sites, or classes of sites, on APC were found to be important to the kinetic effects observed. For GDAPC, with both monovalent cations investigated, a single cation-binding site, or class of sites, of kinetic importance was discovered. The most general mechanism that fits all kinetic data was a rapid equilibrium type, with the cation(s) (A) and substrate (S) binding to the enzyme in a random fashion. Cations were found to be essential activators, and only formation of the EAS or EA2S complex led to product generation. For each enzyme, stimulation of the reaction rates was found to be chiefly due to a dramatic enhancement by monovalent cations of the rate constant (k2) for acylation of the enzyme since the dissociation constant (Ks) for enzyme-substrate interactions was increased in the presence of cations, and the deacylation rate constant (k3) was not affected by these activators.     

Comparison of pepsins isolated from porcine, bovine and Penicillium jathiuellum

1. The reactivities of pepsins, isolated from three different sources (porcine, bovine, and Penicillium jathiuelum), toward ester and peptide substrates were compared. 2. Porcine pepsin showed the highest activity followed by penicillopepsin with bovine pepsin being the least active. 3. The esterase activity of penicillopepsin was greater than that of porcine pepsin with bovine pepsin again showing the least activity. 4. The CD spectra indicate that porcine and bovine pepsin have similar conformations, even though bovine pepsin shows less ellipticity at 220 nm. 5. Penicillopepsin showed a completely opposite sign in the near-u.v. region of the CD spectrum. 6. The far-u.v. region of the CD spectrum of penicillopepsin strongly suggests a beta-sheet structure. 7. Previously reported X-ray crystallographic data suggest that porcine pepsin has a compact three-dimensional structure, while the structures of bovine and penicillopepsins are partially unfolded.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

An estrogen-dependent esterase activity in MCF-7 cells

The presence of steroidal esters in hormonally sensitive tissues lends importance to the esterases which convert the biologically inactive adducts to the parent potent forms. Accordingly, esterase-activities were studied in a human breast cancer model--the MCF-7 cell line. Tritiated estradiol esters- estradiol-17-acetate (EA), estradiol-17-valerate (EV) and estradiol-17-stearate (ES) were tested systematically, but 3 beta-ol esters of androgens, and phorbol diesters were also investigated. All compounds tested, except the phorbol diesters were hydrolyzed either when added to growing cultures or to the 28,000 g supernate of homogenized MCF-7 cells. Among the estrogens, the relative rates of hydrolysis were EA greater than EV greater than ES. The esterase for EA was different as it was not inhibited by saturating concentrations of EV or ES, and unlike the others its activity was stimulated by the addition of estradiol to the culture medium. The antiestrogen keoxifene,[(6-Hydroxy-2-(4-hydroxyphenyl)benzo less than b greater than thien-3-yl greater than less than 4- less than 2-(1-piperidinyl)ethoxy greater than phenyl greater than methanone], negated the stimulatory effect. Other major classes of steroids did not influence EA esterase activity. Results of inhibition experiments indicated that the esterases are of the serine active-site types. The significance of the estrogen-dependent esterase activity can be assessed when the natural substrate(s) for the enzyme is elucidated.     

Purification and characterization of bovine pancreatic bile salt-activated lipase

An enzyme with lipase and esterase activity was purified from bovine pancreas. Furthermore, a non-radioactive lipase assay was developed which is 100 times more sensitive than the conventional methods and allowed the characterization of the lipase activity of the enzyme. The lipase activity increased 42 times in the presence of 10 mM sodium taurocholate, which for the first time provides direct evidence that a bile salt-activated lipase (bp-BAL) was isolated from bovine pancreas. This conclusion is further supported by the fact that the N-terminal amino acid sequence of this lipase/esterase is 88% homologous to human milk BAL and human pancreatic BAL. Staining with various lectins showed that bp-BAL is a glycoprotein which contains fucose residues. Previously from bovine pancreas a lysophospholipase has been purified and a gene was cloned and sequenced encoding an enzyme with cholesterol esterase/lysophospholipase activity. Comparison of the N-terminal amino acid sequence of bp-BAL with the deduced amino acid sequence of the latter revealed that they are identical. Furthermore, the molecular weight of the purified bp-BAL of 63,000, as estimated by SDS-PAGE, is very similar to that of the purified lysophospholipase (65,000) and to the theoretical molecular weight of 65,147 of the cholesterol esterase/lysophospholipase. These data suggest that these three enzymes are one and the same.     

Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases

Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.     

Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints

Electroacupuncture (EA) at P5-P6 acupoints overlying the median nerve reduces premotor sympathetic cardiovascular neuronal activity in the rostral ventral lateral medulla (rVLM) and visceral reflex pressor responses. In previous studies, we have noted different durations of influence of EA comparing P5-P6 and S36-S37 acupoints, suggesting that point specificity may exist. The purpose of this study was to evaluate the influence of stimulating P5-P6 (overlying the median nerve), LI4-L7 (overlying branches of the median nerve and the superficial radial nerve), LI6-LI7 (overlying the superficial radial nerve), LI10-LI11 (overlying the deep radial nerves), S36-S37 (overlying the deep peroneal nerves), or K1-B67 (overlying terminal branches of the tibial nerves) specific acupoints, overlying deep and superficial somatic nerves, on the excitatory cardiovascular reflex and rVLM responses evoked by stimulation of chemosensitive receptors in the cat's gallbladder with bradykinin (BK) or direct splanchnic nerve (SN) stimulation. We observed point-specific differences in magnitude and duration of EA inhibition between P5-P6 or LI10-LI11 and LI4-L7 or S36-S37 in responses to 30-min stimulation with low-frequency, low-current EA. EA at LI6-LI7 and K1-B67 acupoints as well as direct stimulation of the superficial radial nerve did not cause any cardiovascular or rVLM neuronal effects. Cardiovascular neurons in the rVLM, a subset of which were classified as premotor sympathetic cells, responded to brief (30 s) stimulation of the SN as well as acupoints P5-P6, LI10-LI11, LI4-L7, S36-S37, LI6-LI7, or K1-B67, or underlying somatic pathways in a fashion similar to the reflex responses. In fact, we observed a significant linear relationship (r(2) = 0.71) between the evoked rVLM response and reflex change in mean arterial blood pressure. In addition, EA stimulation at P5-P6 and LI4-L7 decreased rVLM neuronal activity by 41 and 12%, respectively, for >1 h, demonstrating that prolonged input into the medulla during stimulation of somatic nerves, depending on the degree of convergence, leads to more or less inhibition of activity of these cardiovascular neurons. Thus EA at acupoints overlying deep and superficial somatic nerves leads to point-specific effects on cardiovascular reflex responses. In a similar manner, sympathetic cardiovascular rVLM neurons that respond to both visceral (reflex) and somatic (EA) nerve stimulation manifest graded responses during stimulation of specific acupoints, suggesting that this medullary region plays a role in site-specific inhibition of cardiovascular reflex responses by acupuncture.     

Measurement of the metabolism of energy substrates in individual bovine blastocysts

Individual blastocysts from cows were cultured for 3 h under 5% CO2 in air, in 4 microliters droplets of Ham's F-10 medium containing D-[5-3H]glucose, D-[1-14C]-glucose, D-[6-14C]glucose, [2-14C]pyruvate, or L-[U-14C]glutamine, and with or without 2,4-dinitrophenol (DNP) or phenazine ethosulphate (PES). The 14CO2 or 3H2O produced were collected by exchange with an outer bath of 400 microliter 25 mM-NaHCO3. All combinations of substrate and treatment (control, DNP or PES) produced measurable quantities of labelled product except for D-[6-14C]glucose in the presence of PES. Untreated and DNP-treated embryos developed normally during a subsequent 48-h culture period in fresh medium, but PES-treated embryos degenerated. Pyruvate and glutamine metabolism both increased markedly in the presence of DNP, indicating that the Krebs' cycle is active, and that glutamine can be used as an energy substrate. Conversely, DNP has no significant effect on glucose metabolism, indicating that glycolysis is blocked in the bovine blastocyst due to a lack or inhibition of pyruvate kinase. The production of 14CO2 from D-[1-14C]glucose increased significantly in the presence of PES, indicating that the activity of the pentose shunt is less than maximal.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart.

CuCl2 non-competitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg2+, 10 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl2 was not additive with that of p-hydroxymercuribenzoate, therefore CuCl2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I50 value of 18 muM. Several effects of Cu2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Effect of electroacupuncture on the cervicospinal P2X7 receptor/fractalkine/CX3CR1 signaling pathway in a rat neck-incision pain model

Increasing evidence supports that acupuncture intervention is an effective approach for intraoperative and postoperative pain. Neuron–microglia crosstalk, mediated by the purinergic P2X7 receptor (R)/fractalkine/CX3CR1 cascade in the spinal cord dorsal horn, plays a pivotal role in pain processing. However, its involvement in the analgesic effect of electroacupuncture (EA) remains unclear. In this study, a rat neck-incision pain model was established by making a longitudinal incision along the midline of the neck and subsequent repeated mechanical stimulation. EA stimulation was applied to bilateral LI18, LI4-PC6, or ST36-GB34. The thermal pain threshold, cervicospinal ATP concentration, expression levels of purinergic P2XR and P2YR subunits mRNAs, and fractalkine, CX3CR1 and p38 MAPK proteins, were detected separately. The neck incision induced strong thermal hyperalgesia and upregulation of spinal ATP within 48 h. No significant change was found in thermal hyperalgesia after a single session of EA intervention. However, a single session of EA dramatically enhanced the neck incision-induced upregulation of ATP and upregulated the expression of P2X7R, which was reversed by two sessions of EA. Two sessions of EA at bilateral LI18 or LI4-PC6 attenuated hyperalgesia significantly, accompanied with downregulation of P2X7R/fractalkine/ CX3CR1 signaling after three sessions of EA. EA stimulation of LI18 or LI4-PC6 alleviates thermal hyperalgesia in neck-incision pain rats, which may be associated with its effects in regulating the neck incision-induced increase of ATP and P2X7R and subsequently suppressing fractalkine/CX3CR1 signaling in the cervical spinal cord.     

Purification and properties of RNA polymerase of phage KB1

Bacteriophage KB1 belongs to group C of Bradely's classification After infection a bacteriophage specific RNA polymerase is induced in infected cells. KB1 RNA polymerase is a stable enzyme and is easily purified to homogeneity in good overall yield. The activity resides in a single polypeptide chain of molecular weight about 90,000. Synthesis of RNA by KB1 RNA polymerase requires a DNA template and Mg++ and like SP6 RNA polymerase, is strongly stimulated by either bovine serum albumin or spermidine. Thiol reactive reagents inhibit the enzyme, suggesting the presence of essential sulfhydryl residues. The enzyme possess a stringent promoter specificity. The KB1 RNA polymerase is also highly active in synthesis of poly(rG) with poly(dI).(dC) as template. My experiments suggest that the catalytic portion of the polymerase can be separated from the RNA polymerase holoenzyme.     

Purification and Characterization of a Lovastatin Esterase from Clonostachys compactiuscula

An esterase from the fungus Clonostachys compactiuscula selectively hydrolyzes lovastatin, a clinically useful antihypercholesterolemic agent. Lovastatin or lovastatin-related compounds were required to induce the activity of the lovastatin 8(prm1)-((alpha)-methylbutyryloxy) esterase. The 46-kDa esterase was purified from mycelial extracts by centrifugation and a single anion-exchange chromatographic separation. Maximal lovastatin esterase activity was found at pH 9.0 to 9.6 and at 25 to 30(deg)C. The addition of 5 to 20% methanol resulted in greater lovastatin hydrolysis, while the addition of other solvents (ethanol, isopropanol, butanol, ethyl acetate, isopropyl acetate, or tetrahydrofuran) decreased hydrolysis. Lovastatin was selectively hydrolyzed even in the presence of an excess of simvastatin, another antihypercholesterolemic agent that is structurally very similar to lovastatin. This lovastatin 8(prm1)-((alpha)-methylbutyryloxy) esterase can be used to prepare a core intermediate for the generation of novel antihypercholesterolemic agents or to purify simvastatin prepared by C methylation of the 2(S)-methylbutyryloxy side chain of lovastatin.     

Influenza C virus and bovine coronavirus esterase reveal a similar catalytic mechanism: new insights for drug discovery

Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2))-containing glycans. Here we report nuclear magnetic resonance and molecular modelling studies on the 9-O-acetyl esterase showing that the alpha-configured Neu5,9Ac(2) is strictly preferred by the INF-C and BCoV esterases. Interestingly, we have discovered that the INF-C esterase function releases acetate independently of the chemical nature of the aglycon moiety, whereas subtle differences in substrate recognition were found for BCoV esterase. Analysis of the apo and complexed X-ray crystal structure of INF-C esterase revealed that binding of 9-O-acetylated N-acetylneuraminic acids is a dynamic process that involves conformational rearrangement of serine-57 in the esterase active site. This study provides valuable insights towards the design of drugs to combat INF-C virus and coronavirus infections causing outbreaks of upper respiratory infections and severe diarrhea in calves, respectively.