Differentiating sepsis from non-infectious systemic inflammation based on microvesicle-bacteria aggregation.

Sepsis is a severe medical condition and a leading cause of hospital mortality. Prompt diagnosis and early treatment has a significant, positive impact on patient outcome. However, sepsis is not always easy to diagnose, especially in critically ill patients. Here, we present a conceptionally new approach for the rapid diagnostic differentiation of sepsis from non-septic intensive care unit patients. Using advanced microscopy and spectroscopy techniques, we measure infection-specific changes in the activity of nano-sized cell-derived microvesicles to bind bacteria. We report on the use of a point-of-care-compatible microfluidic chip to measure microvesicle-bacteria aggregation and demonstrate rapid (≤1.5 hour) and reliable diagnostic differentiation of bacterial infection from non-infectious inflammation in a double-blind pilot study. Our study demonstrates the potential of microvesicle activities for sepsis diagnosis and introduces microvesicle-bacteria aggregation as a potentially useful parameter for making early clinical management decisions.

Effect of increasing the protein-to-fat ratio and reducing fat content on the chemical and physical properties of processed cheese product.

Scientific studies indicate that the intake of dietary fat and saturated fats in the modern Western diet is excessive and contributes adversely to health, lifestyle, and longevity. In response, manufacturers of cheese and processed cheese products (PCP) are pursuing the development of products with reduced fat contents. The present study investigated the effect of altering the fat level (13.8, 18.2, 22.7, 27.9, and 32.5 g/100g) in PCP on their chemical and physical properties. The PCP were formulated in triplicate to different fat levels using Cheddar cheese, skim milk cheese, anhydrous milk fat, emulsifying salt (ES), NaCl, and water. The formulations were designed to give fixed moisture (~53 g/100g) and ES:protein ratio (0.105). The resultant PCP, and their water-soluble extracts (WSE), prepared from a macerated blend of PCP and water at a weight ratio of 1:2, were analyzed at 4d. Reducing the fat content significantly increased the firmness of the unheated PCP and reduced the flowability and maximum loss tangent (fluidity) of the melted PCP. These changes coincided with increases in the levels of total protein, water-soluble protein, water-insoluble protein, and water-soluble Ca, and a decrease in the molar ratio of water-soluble Ca to soluble P. However, both water-soluble Ca and water-soluble protein decreased when expressed as percentages of total protein and total Ca, respectively, in the PCP. The high level of protein was a major factor contributing to the deterioration in physical properties as the fat content of PCP was reduced. Diluting the protein content or reducing the potential of the protein to aggregate, and thereby form structures that contribute to rigidity, may provide a means for improving quality of reduced-fat PCP by using natural cheese with lower intact casein content and lower calcium:casein ratio, for example, or by decreasing the ratio of sodium phosphate to sodium citrate-based ES.

Effects of hydrolysis on solid-state relaxation and stickiness behavior of sodium caseinate-lactose powders.

Hydrolyzed or nonhydrolyzed sodium caseinate-lactose dispersions were spray dried, at a protein: lactose ratio of 0.5, to examine the effects of protein hydrolysis on relaxation behavior and stickiness of model powders. Sodium caseinate (NC) used included a nonhydrolyzed control (DH 0) and 2 hydrolyzed variants (DH 8.3 and DH 15), where DH = degree of hydrolysis (%). Prior to spray drying, apparent viscosities of liquid feeds (at 70°C) at a shear rate of 20/s were 37.6, 3.14, and 3.19 mPa·s, respectively, for DH 0, DH 8, and DH 15 dispersions. Powders containing hydrolyzed casein were more susceptible to sticking than those containing intact NC. The former had also lower bulk densities and powder particle sizes. Scanning electron microscopy showed that hydrolyzed powders had thinner particle walls and were more friable than powders containing intact NC. Secondary structure of caseinates, determined by Fourier transform infrared spectroscopy, was affected by the relative humidity of storage and the presence of lactose as co-solvent rather than its physical state. Glass transition temperatures and lactose crystallization temperatures, determined by differential scanning calorimetry were not affected by caseinate hydrolysis, although the effects of protein hydrolysis on glass-rubber transitions (T(gr)) could be determined by thermo-mechanical analysis. Powders containing hydrolyzed NC had lower T(gr) values (~30°C) following storage at a higher subcrystallization relative humidity (33%) compared with powder with nonhydrolyzed NC (T(gr) value of ~40°C), an effect that reflects more extensive plasticization of powder matrices by moisture. Results support that sodium caseinate-lactose interactions were weak but that relaxation behavior, as determined by the susceptibility of powder to sticking, was affected by hydrolysis of sodium caseinate.

Preliminary evaluation of endogenous milk fluorophores as tracer molecules for curd syneresis.

A front-face fluorescence spectroscopy probe was installed in the wall of a laboratory-scale cheese vat. Excitation and emission filters were chosen for the selective detection of vitamin A, tryptophan, and riboflavin fluorescence. The evolution of the fluorescence of each fluorophore during milk coagulation and syneresis was monitored to determine if they had the potential to act as intrinsic tracers of syneresis and also coagulation. The fluorescence profiles for 2 of the fluorophores during coagulation could be divided into 3 sections relating to enzymatic hydrolysis of κ-casein, aggregation of casein micelles, and crosslinking. A parameter relating to coagulation kinetics was derived from the tryptophan and riboflavin profiles but this was not possible for the vitamin A response. The study also indicated that tryptophan and riboflavin may act as tracer molecules for syneresis, but this was not shown for vitamin A. The evolution of tryptophan and riboflavin fluorescence during syneresis followed a first-order reaction and had strong relationships with curd moisture and whey total solids content (r=0.86-0.96). Simple 1- and 2-parameter models were developed to predict curd moisture content, curd yield, and whey total solids using parameters derived from the sensor profiles (standard error of prediction=0.0005-0.394%; R(2)=0.963-0.999). The results of this study highlight the potential of tryptophan and riboflavin to act as intrinsic tracer molecules for noninvasive inline monitoring of milk coagulation and curd syneresis. Further work is required to validate these findings under a wider range of processing conditions.

Effects of milk composition, stir-out time, and pressing duration on curd moisture and yield.

A study was undertaken to investigate the effects of milk composition (i.e., protein level and protein:fat ratio), stir-out time, and pressing duration on curd moisture and yield. Milks of varying protein levels and protein:fat ratios were renneted under normal commercial conditions in a pilot-scale cheese vat. During the syneresis phase of cheese making, curd was removed at differing times, and curd moisture and yield were monitored over a 22-h pressing period. Curd moisture after pressing decreased with longer stir-out time and pressing duration, and an interactive effect was observed of stir-out time and pressing duration on curd moisture and yield. Milk total solids were shown to affect curd moisture after pressing, which has implications for milk standardization; that is, it indicates a need to standardize on a milk solids basis as well as on a protein:fat basis. In this study, a decreased protein:fat ratio was associated with increased total solids in milk and resulted in decreased curd moisture and increased curd yield after pressing. The variation in total solids of the milk explains the apparent contradiction between decreased curd moisture and increased curd yield. This study points to a role for process analytic technology in minimizing variation in cheese characteristics through better control of cheesemilk composition, in-vat process monitoring (coagulation and syneresis), and post-vat moisture reduction (curd pressing). Increased control of curd composition at draining would facilitate increased control of the final cheese grade and quality.

Comparison between red-green-blue imaging and visible-near infrared reflectance as potential process analytical tools for monitoring syneresis.

The current work focuses on the comparison of 2 on-line optical sensing systems; namely red-green-blue imaging and visible-near infrared reflectance, for monitoring syneresis during cheese manufacture. The experimental design consisted of 3 temperature treatments carried out in an 11-L cheese vat in triplicate. Both systems were shown to predict syneresis without significant differences in prediction accuracy. However, a single-wavelength near infrared model was the most parsimonious (standard error of prediction=4.35g/100g) for predicting syneresis. This technique was also the simplest in terms of parameters in the model (standard error of prediction=4.15g/100g with 2 parameters), when time after gel cutting and process parameters (temperature and cutting time) were included in the models. The study showed that either system could be employed to control syneresis in cheese manufacture and improve the control of moisture content in cheese.

Validation of a curd-syneresis sensor over a range of milk composition and process parameters.

An online visible-near-infrared sensor was used to monitor the course of syneresis during cheesemaking with the purpose of validating syneresis indices obtained using partial least squares, with cross-validation across a range of milk fat levels, gel firmness levels at cutting, curd cutting programs, stirring speeds, milk protein levels, and fat:protein ratio levels. Three series of trials were carried out in an 11-L cheese vat using recombined whole milk. Three factorial experimental designs were used, consisting of 1) 3 curd stirring speeds and 3 cutting programs; 2) 3 milk fat levels and 3 gel firmness levels at cutting; and 3) 2 milk protein levels and 3 fat:protein ratio levels, respectively. Milk was clotted under constant conditions in all experiments and the gel was cut according to the respective experimental design. Prediction models for production of whey and whey fat losses were developed in 2 of the experiments and validated in the other experiment. The best models gave standard error of prediction values of 6.6 g/100 g for yield of whey and 0.05 g/100 g for fat in whey, as compared with 4.4 and 0.013 g/100 g, respectively, for the calibration data sets. Robust models developed for predicting yield of whey and whey fat losses using a validation method have potential application in the cheese industry.

Effects of cutting intensity and stirring speed on syneresis and curd losses during cheese manufacture.

Recombined whole milk was renneted under constant conditions of pH, temperature, and added calcium, and the gel was cut at a constant firmness. The effects of cutting and stirring on syneresis and curd losses to whey were investigated during cheese making using a factorial design with 3 cutting modes designed to provide 3 different cutting intensity levels (i.e., total cutting revolutions), 3 levels of stirring speed, and 3 replications. These cutting intensities and stirring speeds were selected to give a wide range of curd grain sizes and curd shattering, respectively. Both factors affected curd losses, and correct selection of these factors is important in the cheesemaking industry. Decreased cutting intensity and increased stirring speed significantly increased the losses of fines and fat from the curd to the whey. Cutting intensities and stirring speeds in this study did not show significant effects on curd moisture content over the course of syneresis. Levels of total solids, fines, and fat in whey were shown to change significantly during syneresis. It is believed that larger curd particles resulting from low cutting intensities coupled with faster stirring speeds resulted in a higher degree of curd shattering during stirring, which caused significant curd losses.

A randomized, double-blind, crossover comparison of the efficacy and safety of oral controlled-release tramadol and placebo in patients with painful osteoarthritis.

OBJECTIVE: To compare the efficacy and safety of controlled-release (CR) tramadol (Zytram XL, Purdue Pharma, Canada) and placebo in patients with painful osteoarthritis. METHODS: Patients underwent analgesic washout for two to seven days before random assignment to 150 mg daily of CR tramadol or placebo, and were titrated weekly to 200 mg, 300 mg or a maximum of 400 mg once daily. After four weeks, patients crossed over to the alternate treatment for another four weeks. Plain acetaminophen was provided as a rescue analgesic. All patients who completed the crossover study were eligible to receive open label CR tramadol for six months. RESULTS: Seventy-seven of 100 randomly assigned patients were evaluable for efficacy. CR tramadol resulted in significantly lower visual analogue scale pain intensity scores (37.4+/-23.9 versus 45.1+/-24.3, P=0.0009). Western Ontario and McMaster Universities osteoarthritis index subscale scores for pain (189.0+/-105.0 versus 230.0+/-115.4; P=0.0001) and physical function (632.4+/-361.3 versus 727.4+/-383.4; P=0.0205) were significantly better with CR tramadol. Total pain and disability (22.8+/-14.5 versus 27.2+/-14.8; P=0.0004), and overall pain and sleep (104.7+/-98.0 versus 141.0+/-108.2; P=0.0005) scores in the Pain and Sleep Questionnaire were significantly lower for CR tramadol. Short-form 36 Health Survey scores were significantly better during CR tramadol treatment for the pain index (38.8+/-10.8 versus 35.6+/-9.0; P=0.0100), general health perception (46.5+/-11.2 versus 44.4+/-11.6; P=0.0262), vitality (43.1+/-13.2 versus 40.2+/-13.7; P=0.0255) and overall physical components (40.8+/-8.9 versus 37.8+/-7.7; P=0.0002). CR tramadol treatment was preferred by 55.8% of patients (P=0.0005) versus 20.8% and 23.4% of patients who chose placebo or had no preference, respectively. These improvements were sustained for up to six months, and 86.5% of patients reported at least moderate benefit from CR tramadol during long-term treatment. CONCLUSION: CR tramadol is effective for the management of painful osteoarthritis.

Application of image texture analysis for online determination of curd moisture and whey solids in a laboratory-scale stirred cheese vat.

A noninvasive technology, which could be employed online to monitor syneresis, would facilitate the production of higher quality and more consistent cheese products. Computer vision techniques such as image texture analysis have been successfully established as rapid, consistent, and nondestructive tools for determining the quality of food products. In this study, the potential of image texture analysis to monitor syneresis of cheese curd in a stirred vat was studied. A fully randomized 2-factor (milk pH and stirring speed), 2-level factorial design was carried out in triplicate. During syneresis, images of the surface of the stirred curd-whey mixture were captured using a computer vision system. The images were subjected to 5 image texture analysis methods by which 109 image texture features were extracted. Significant correlations were observed between a number of image texture features and curd moisture and whey solids. Multiscale analysis techniques of fractal dimension and wavelet transform were demonstrated to be the most useful for predicting syneresis indices. Fractal dimension features predicted curd moisture and whey solids during syneresis with standard errors of prediction of 1.03% (w/w) and 0.58 g/kg, respectively. It was concluded that syneresis indices were most closely related to the image texture features of multiscale representation. The results of this study indicate that image texture analysis has potential for monitoring syneresis.

Application of mid-infrared spectroscopy to the prediction of maturity and sensory texture attributes of cheddar cheese.

The objective of this study was to determine the potential of mid-infrared spectroscopy in conjunction with partial least squares (PLS) regression to predict various quality parameters in cheddar cheese. Cheddar cheeses (n= 24) were manufactured and stored at 8 degrees C for 12 mo. Mid-infrared spectra (640 to 4000/cm) were recorded after 4, 6, 9, and 12 mo storage. At 4, 6, and 9 mo, the water-soluble nitrogen (WSN) content of the samples was determined and the samples were also evaluated for 11 sensory texture attributes using descriptive sensory analysis. The mid-infrared spectra were subjected to a number of pretreatments, and predictive models were developed for all parameters. Age was predicted using scatter-corrected, 1st derivative spectra with a root mean square error of cross-validation (RMSECV) of 1 mo, while WSN was predicted using 1st derivative spectra (RMSECV = 2.6%). The sensory texture attributes most successfully predicted were rubbery, crumbly, chewy, and massforming. These attributes were modeled using 2nd derivative spectra and had corresponding RMSECV values in the range of 2.5 to 4.2 on a scale of 0 to 100. It was concluded that mid-infrared spectroscopy has the potential to predict age, WSN, and several sensory texture attributes of cheddar cheese.

Effect of cutting time, temperature, and calcium on curd moisture, whey fat losses, and curd yield by response surface methodology.

Response surface methodology was used to study the effect of temperature, cutting time, and calcium chloride addition level on curd moisture content, whey fat losses, and curd yield. Coagulation and syneresis were continuously monitored using 2 optical sensors detecting light backscatter. The effect of the factors on the sensors' response was also examined. Retention of fat during cheese making was found to be a function of cutting time and temperature, whereas curd yield was found to be a function of those 2 factors and the level of calcium chloride addition. The main effect of temperature on curd moisture was to increase the rate at which whey was expelled. Temperature and calcium chloride addition level were also found to affect the light backscatter profile during coagulation whereas the light backscatter profile during syneresis was a function of temperature and cutting time. The results of this study suggest that there is an optimum firmness at which the gel should be cut to achieve maximum retention of fat and an optimum curd moisture content to maximize product yield and quality. It was determined that to maximize curd yield and quality, it is necessary to maximize firmness while avoiding rapid coarsening of the gel network and microsyneresis. These results could contribute to the optimization of the cheese-making process.

Computer vision and color measurement techniques for inline monitoring of cheese curd syneresis.

Optical characteristics of stirred curd were simultaneously monitored during syneresis in a 10-L cheese vat using computer vision and colorimetric measurements. Curd syneresis kinetic conditions were varied using 2 levels of milk pH (6.0 and 6.5) and 2 agitation speeds (12.1 and 27.2 rpm). Measured optical parameters were compared with gravimetric measurements of syneresis, taken simultaneously. The results showed that computer vision and colorimeter measurements have potential for monitoring syneresis. The 2 different phases, curd and whey, were distinguished by means of color differences. As syneresis progressed, the backscattered light became increasingly yellow in hue for circa 20 min for the higher stirring speed and circa 30 min for the lower stirring speed. Syneresis-related gravimetric measurements of importance to cheese making (e.g., curd moisture content, total solids in whey, and yield of whey) correlated significantly with computer vision and colorimetric measurements.

Evaluating mid-infrared spectroscopy as a new technique for predicting sensory texture attributes of processed cheese.

The objective of this study was to investigate the potential application of mid-infrared spectroscopy for determination of selected sensory attributes in a range of experimentally manufactured processed cheese samples. This study also evaluates mid-infrared spectroscopy against other recently proposed techniques for predicting sensory texture attributes. Processed cheeses (n = 32) of varying compositions were manufactured on a pilot scale. After 2 and 4 wk of storage at 4 degrees C, mid-infrared spectra (640 to 4,000 cm(-1)) were recorded and samples were scored on a scale of 0 to 100 for 9 attributes using descriptive sensory analysis. Models were developed by partial least squares regression using raw and pretreated spectra. The mouth-coating and mass-forming models were improved by using a reduced spectral range (930 to 1,767 cm(-1)). The remaining attributes were most successfully modeled using a combined range (930 to 1,767 cm(-1) and 2,839 to 4,000 cm(-1)). The root mean square errors of cross-validation for the models were 7.4 (firmness; range 65.3), 4.6 (rubbery; range 41.7), 7.1 (creamy; range 60.9), 5.1 (chewy; range 43.3), 5.2 (mouth-coating; range 37.4), 5.3 (fragmentable; range 51.0), 7.4 (melting; range 69.3), and 3.1 (mass-forming; range 23.6). These models had a good practical utility. Model accuracy ranged from approximate quantitative predictions to excellent predictions (range error ratio = 9.6). In general, the models compared favorably with previously reported instrumental texture models and near-infrared models, although the creamy, chewy, and melting models were slightly weaker than the previously reported near-infrared models. We concluded that mid-infrared spectroscopy could be successfully used for the nondestructive and objective assessment of processed cheese sensory quality.

Effect of protein-to-fat ratio of milk on the composition, manufacturing efficiency, and yield of cheddar cheese.

Twenty-three Cheddar cheeses were prepared from milks with a protein content of 3.66% (wt/wt) and with different protein-to-fat ratio (PFR) in the range 0.70 to 1.15; the PFR of each milk differed by 0.02. For statistical analysis, the 23 cheeses were divided into 3 PFR groups: low (LPFR; 0.70 to 0.85), medium (MPFR; 0.88 to 1.00) and high (HPFR; 1.01 to 1.15), which were compared using ANOVA. The numbers of PFR values in the LPFR, MPFR, and HPFR groups were 9, 7, and 7, respectively. Data were also analyzed by linear regression analysis to establish potentially significant relationships among the PFR and response variables. Increasing PFR significantly increased the levels of cheese moisture, protein, Ca, and P, but significantly reduced the levels of moisture in nonfat substances, fat-in-DM, and salt-in-moisture. The percentage of milk fat recovered in the LPFR cheese was significantly lower than that in the MPFR or HPFR cheeses. In contrast, the recovery of water from milk to the LPFR cheese was significantly higher than that in the MPFR or HPFR cheeses. Increasing the PFR led to a significant decrease in the actual yield of cheese per 100 kg of milk but a significant increase occurred in the normalized yield of cheese per 100 kg of milk with reference values of fat plus protein (3.4 and 3.3%, wt/wt, respectively). The results demonstrate that alteration of the PFR of cheese milk in the range 0.70 to 1.15 has marked effects on cheese composition, component recoveries, and cheese yield.

Cloning of the genomes of equine herpesvirus type 1 (EHV-1) strains KyA and racL11 as bacterial artificial chromosomes (BAC).

The genome of equine herpesvirus type 1 (EHV-1) strain RacL11, a highly virulent isolate obtained from an aborted foal, and that of the modified live vaccine strain KyA, were cloned as bacterial artificial chromosomes (BAC) in Eseherichia coli. Mini F plasmid sequences were inserted into the viral genomes by homologous recombination instead of the gene 71 (EUS4) open reading frame after co-transfection of viral DNA and recombinant plasmid pdelta71-pHA2 into RK13 cells. After isolation of recombinant viruses by three rounds of plaque purification, viral DNA was isolated from RK13 cells infected with RacL11 or KyA virus mutants expressing the green fluorescent protein (GFP), and electroporated into Escherichia coli DH10B cells. Several bacterial colonies were shown to contain high-molecular weight BAC DNA with a restriction enzyme fragment pattern indicative of the presence of full-length RacL11 or KyA genomes. Two selected BAC clones were analysed by restriction enzyme analysis and Southern blotting, and were eventually termed pRacLI I and pKyA. respectively. Upon transfection of pRacL11 or pKyA DNA into RK13 cells, GFP-expressing fluorescing virus plaques could be identified from day 1 after transfection. Infectivity after transfection of pRacL11 or pKyA could be readily propagated on RK13 or equine cells, indicating that infectious full-length DNA clones of strains RacL11 and KyA were successfully cloned in Escherichia coli as BACs. The glycoprotein 2-negative progeny reconstituted from pRacL11 and pKyA (L11deltagp2 and KyAdeltagp2) exhibited different growth properties. Whereas both L11deltagp2 and KyAdeltagp2 extracellular titres were reduced by 15- to 32-fold, plaque diameters were only markedly (50%) reduced in the case of KyAdeltagp2.

Mapping the sequences that mediate interaction of the equine herpesvirus 1 immediate-early protein and human TFIIB.

The sole immediate-early (IE) gene of equine herpesvirus 1 encodes a 1,487-amino-acid (aa) regulatory phosphoprotein that independently activates expression of early viral genes. Coimmunoprecipitation assays demonstrated that the IE protein physically interacts with the general transcription factor TFIIB. Using a variety of protein-binding assays that employed a panel of IE truncation and deletion mutants expressed as in vitro-synthesized or glutathione S-transferase fusion proteins, we mapped a TFIIB-binding domain to aa 407 to 757 of the IE protein. IE mutants carrying internal deletions of aa 426 to 578 and 621 to 757 were partially defective for TFIIB binding, indicating that aa 407 to 757 may harbor more than one TFIIB-binding domain. The interaction between the IE protein and TFIIB is of physiological importance, as evidenced by transient-cotransfection assays. Partial deletion of the TFIIB-binding domain within the IE protein inhibited its ability to activate expression of the viral thymidine kinase gene, a representative early promoter, and of the IR5 gene, a representative late promoter, by greater than 20 and 50%, respectively. These results indicate that the interaction of the IE protein with TFIIB is necessary for its full transactivation function and that the IE-TFIIB interaction may be part of the mechanism by which the IE protein activates transcription.

Molecular characterizations of the equine herpesvirus 1 ETIF promoter region and translation initiation site.

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alphaTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12), was shown to trans-activate immediate-early (IE) gene promoters, and was described as a 60-kDa virion component designated ETIF. However, the ETIF promoter region and transcription initiation site were not identified. The poly(A) signal of the gene 11 (UL49 homolog) lies just upstream of the first ETIF translation initiation codon, indicating that the first ATG may not be used for initiating ETIF translation. Another in-frame translation initiation codon (ATG2) is located 88 bp downstream of the first ETIF initiation codon (ATG1). Western blot analysis showed that the expressed ETIF protein migrated in SDS-PAGE with an apparent molecular mass of approximately 56 kDa, the same molecular weight identified in SDS-PAGE analysis of the KyD EHV-1 virion preparations. The ETIF expression vector pCETIF, which contains ATG2, trans-activated the IE promoter more efficiently than the pC12 containing both ATG1 and ATG2. S1 nuclease analyses mapped the 5' initiation site of the 1.4-kb transcript approximately 17 to 21 nt downstream of the ATG1. The nucleotide sequence upstream of the ATG1 did not have any promoter activity, while the nucleotide sequence upstream of the ATG2 had promoter activity. In transient transfection assays, the pETIFM2 vector, which was mutated in the ATG2, did not trans-activate the IE promoter; however, the pETIFM1 vector, which was mutated in the ATG1, trans-activated the IE promoter. These results demonstrated that the ATG2 of the ETIF ORF is the ETIF translation initiation codon. ETIF trans-activated only the IE promoter, not early (EICP0, EICP22, EICP27, and thymidine kinase) or late (IR5) promoters, confirming that EICP0, EICP22, and EICP27 are early genes.