Life-saving transfusion in autoimmune hemolytic anemia: a case report and procedure review of the dilution method.

A woman with autoimmune hemolytic anemia (AIHA) presented in the emergency department with life-threatening anemia (hemoglobin 3 g/dL). Exaggeration of preexisting chronic anemia to severe anemia after a recent red blood cell (RBC) transfusion led to suspicion of delayed hemolytic transfusion reaction. Given the urgency for transfusion along with a stronger suspicion for coexistence of an alloantibody, the dilution method proposed by Lawrence Petz and George Garratty was used to find an RBC unit for transfusion. An alloantibody with Fyb specificity was identified, which was masked by the coexistent autoantibody. This method is based on the assumption that the titers of an alloantibody are higher than that of autoantibody. Diluting the autoantibody would reveal the alloantibody and, for this purpose, a serial doubling dilution of serum is performed. This method has an important limitation of missing any alloantibodies with titers less than that of the autoantibody. In spite of this, this method may be of use at a resource-poor setting, where trained personnel and other reagents intended for advanced immunohematology methods are unavailable.

A rare case of primary adenoid cystic carcinoma of lung.

A 33-year-old male presented with repeated episodes of blood-streaked sputum for last one-and half-year. Chest radiograph showed consolidation in the right lower zone. Fibreoptic bronchoscopy revealed an endoluminal growth in the right lower lobe bronchus. Histopathological examination of bronchoscopic biopsy specimen confirmed adenoid cystic carcinoma.

Changes in Patterns of Trauma Injuries in the Dakotas Region During the COVID-19 Pandemic.

BACKGROUND: The aim of this study is to compare impact of COVID-19 on trauma volume and characteristics on a set of trauma centers with a rural catchment area. The COVID-19 pandemic has affected different parts of the country quite differently, both in case volume and in local responses. State-wide responses have varied considerably, including variations in local mask mandates, school closures, and social distancing measures. METHODS: This was a retrospective trauma registry review of patients who were admitted to three of the tertiary care trauma centers in North and South Dakota between 2014 through 2022. RESULTS: In the analysis of 36,397 patients, we found a significant increase in trauma patient volume during the COVID-19 pandemic, with an increased percentage of patients presenting with a mechanism of injury secondary to abuse or assault. This increase in patient volume continued to rise during 2021 and 2022. CONCLUSIONS: Our study demonstrates how the COVID-19 pandemic impacted trauma center admissions in the rural and frontier Midwest differently from more urban areas, and the importance of including a variety of settings in trauma research.

Delayed haemolytic transfusion reaction due to Kidd antibodies.

A delayed haemolytic transfusion reaction (DHTR) encompasses a positive direct antiglobulin test (DAT) developed anytime between 24 hours to 28days after cessation of transfusion, a positive eluate or a newly identified alloantibody in the plasma or serum along with features of haemolysis in the patient. Routinely, it is expected that with the transfusion of one unit of packed red cells in a patient of average height and weight, the haemoglobin level and hematocrit increase by 1 g/dL and 3% respectively. However, in a patient with DHTR, an inadequate rise of post-transfusion haemoglobin (<1 g/dL) or rapid fall in haemoglobin back to pre-transfusion levels is observed. Kidd antibodies are particularly known to cause DHTR, maybe alone or in unison with other antibodies. Detection of these alloantibodies is consequential in providing good transfusion support to these patients. These events may be difficult to detect as they may present as varied clinical features or immunological nuisances. In this case series, we have presented three cases of DHTR with special emphasis on their clinical presentations, immunohaematological evaluations, laboratory parameters and the role of proper transfusion support in these patients to avoid further complications.

Physiological and biochemical consequences of host-plasmid interaction--a case study with Corynebacterium renale, a multiple cryptic plasmid containing strain.

Corynebacterium renale harbors four small cryptic plasmids, pCR1, pCR2, pCR3 and pCR4, and can be a good system for understanding host-plasmid interactions. In the present study, effect of plasmid loss and their subsequent introduction on various properties of the host was evaluated. Loss of plasmids caused a reduction in bacterial size and also slowed down their growth rate, µ, and respiratory rate, r. Both µ and r values were partially recovered in C. renale R, obtained by retransformation of the cured strain with all the four cryptic plasmids. Further delineation revealed that a 3153bp plasmid pCR2 alone is sufficient for the observed increase in µ in C. renale R. The advantages conferred by the remaining three plasmids may be are two subtle to be seen under laboratory conditions. Overall, the observations point to the gross metabolic crisis in the host partly as a result of loss of plasmids. Based on the findings, a mutualistic relationship between the host and the plasmids resulting from their coevolution is proposed.

Molecular and biochemical mining of heat-shock and 14-3-3 proteins in drug-induced protoscolices of Echinococcus granulosus and the detection of a candidate gene for anthelmintic resistance.

Cystic echinococcosis (CE) caused by the larval stage of Echinococcus granulosus is a disease that affects both humans and animals. In humans the disease is treated by surgery with a supplementary option of chemotherapy with a benzimidazole compound. During the present study heat-shock protein 60 (HSP 60) was identified as one of the most frequently expressed biomolecules by E. granulosus after albendazole treatment. Data were correlated with 14-3-3 protein signature, and overexpression of this molecule after albendazole induction was an indicator of cell survival and signal transduction during in vitro maintenance of E. granulosus for up to 72 h. This observation was further correlated with a uniform expression pattern of a housekeeping gene (actin II). Out of three ß-tubulin gene isoforms of E. granulosus, ß-tubulin gene isoform 2 showed a conserved point mutation indicative of benzimidazole resistance.

Safety, Tolerability and Pharmacokinetics of Single and Repeat Doses of Vixotrigine in Healthy Volunteers.

Neuropathic pain affects ~ 6.9-10% of the general population and leads to loss of function, anxiety, depression, sleep disturbance, and impaired cognition. Here, we report the safety, tolerability, and pharmacokinetics of a voltage-dependent and use-dependent sodium channel blocker, vixotrigine, currently under investigation for the treatment of neuropathic pain conditions. The randomized, placebo-controlled, phase I clinical trials were split into single ascending dose (SAD) and multiple ascending dose (MAD) studies. Healthy volunteers received oral vixotrigine as either single doses followed by a ≥ 7-day washout period for up to 5 dosing sessions (SAD, n = 30), or repeat doses (once or twice daily) for 14 and 28 days (MAD, n = 51). Adverse events (AEs), maximum observed vixotrigine plasma concentration (Cmax ), area under the concentration-time curve from predose to 24 hours postdose (AUC0-24 ), time to Cmax (Tmax ), and terminal half-life (t1/2), among others, were assessed. Drug-related AEs were reported in 47% and 53% of volunteers in the SAD and MAD studies, respectively, with dizziness as the most commonly reported drug-related AE. SAD results showed that Cmax and AUC increased with dose, Tmax was 1-2 hours, and t1/2 was ~ 11 hours. A twofold increase in accumulation was observed when vixotrigine was taken twice vs. once daily (MAD). Steady-state was achieved from day 5 onward. These data indicate that oral vixotrigine is well-tolerated when administered as single doses up to 825 mg and multiple doses up to 450 mg twice daily.

Development and Feasibility of a Prehabilitation Protocol for Patients Diagnosed with Head and Neck Cancer.

BACKGROUND:  Head and neck (H&N) cancers account for 4% of total cancers diagnosed. However, quality of life (QoL) implications are more severe for this patient population due to the complexity, extent, and deformities resulting from treatment interventions. Principally debilitating complications include diminished functional walking capacity, reduced cervical range of motion (ROM), and scapular strength. An extensive literature search revealed a paucity of evidence utilizing physical therapy assessment and intervention for this population. The purpose of this study was to describe the development and clinical feasibility of a prehabilitation program aimed to thwart these complications for patients diagnosed with H&N cancer.  Methods: Inclusion criteria: male or female, 18+ years, speak and read the English language, ambulate independently, diagnosed with H&N cancer, and scheduled for surgical intervention. Institutional Review Board approval was obtained. Pre- and post-surgical measurements included the six-minute walk test (6MWT), cervical ROM, manual muscle testing for scapular strength, and three questionnaires: physical activity history, health behaviors questionnaire, and the Functional Assessment Cancer Therapy H&N QoL survey.  Results: Three participants were enrolled (two males and one female) all identifying as Caucasian and between 60-90 years of age. Pre- to post-cervical ROM demonstrated decline in extension/bilateral rotation for two of three participants. Two participants demonstrated decreased 6MWT distance while one increased. No participants experienced any adverse effects of the prehabilitation program.  Conclusion: This is the first study protocol to describe a physical therapist-administered H&N cancer prehabilitation program. Professionally administered education and exercise has potential to prevent, manage, and mitigate the adverse effects of cancer treatment. Additional research is needed to define the importance of prehabilitation relative to improved clinical outcomes and improved QoL. Patients with a cancer diagnosis are susceptible to impairments and functional limitations as a result of treatments and this prehabilitation program demonstrates potential to positively impact outcomes across the survivorship continuum. Due to their education and integration within the medical system, physical therapists are well-positioned to lead the effort to unify theory and clearly define parameters for oncology prehabilitation.

Effects of Aerobic Exercise on Oxidative Stress in Patients Diagnosed with Cancer: A Narrative Review.

BACKGROUND: Oxidative stress (OS) can bring about an imbalance between the production of free radicals (pro-oxidants) and their elimination by protective mechanisms (antioxidants). Exercise and/or physical activity (PA) may provide a mechanism to control the variation and equilibrium between pro-oxidants and antioxidants. PURPOSE:  The purpose of this narrative review is to investigate the evidence regarding the effect of exercise and/or PA on OS among individuals diagnosed with cancer.  Methods: A narrative review study design involved a literature search (August 2016) across the databases: Cumulative Index of Nursing and Allied Health Literature (CINAHL), Cochrane, Excerpta Medica database (Embase), and PubMed. Articles included those published from January 2000 - August 2016; inclusive of the search terms "cancer" AND "neoplasm" AND "oncology" AND "oxidative stress" AND "exercise" AND "physical activity"; written in the English language; and utilizing human subjects. The references of the selected articles were then reviewed to identify any qualifying articles. A modified Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) review of each article was completed by two investigators. RESULTS:  Eight articles met the final inclusion criteria. Moderate exercise may provide protective mechanisms against OS via increased antioxidant activity, while exhaustive exercise may be responsible for increased levels of OS, increasing the risk for malignancy. While increased OS levels are utilized by current oncologic therapies to damage malignant and premalignant cells, they also damage healthy cells (cardiac, nerve, and lymphatic). CONCLUSION: Moderate levels of exercise and/or PA may provide preventative and protective qualities against the negative side effects associated with increased OS from cancer treatment.

Implementation and Preliminary Analysis of FACT-G Quality of Life Questionnaire within an Oncology Survivorship Clinic.

PURPOSE: To conduct a descriptive analysis of the results from the Functional Assessment of Cancer Therapy-General (FACT-G) quality of life (QOL) questionnaire, describe the outcomes from the FACT-G to drive treatment recommendations within the breast survivorship clinic and to quantify the severity of QOL issues experienced. METHODS: A retrospective analysis utilizing medical records of participants in a breast cancer survivorship clinic. Measurement data included demographics and FACT-G results. Descriptive analysis of demographics and trends in referral recommendations and FACT-G scores was completed. RESULTS: All 30 participants were females diagnosed with breast cancer of various stages, ages 28 to 81 years. Approximately 1.5 years elapsed between cancer diagnosis and completion of the FACT-G. Participants received surgery (100%), radiation (76%), and chemotherapy and/or hormonal therapy (43%). Results demonstrated that participants reported having a lack of energy (24%) and were bothered by side effects of their treatment (20%). The greatest impact on functional well-being was difficulty sleeping (50%). LIMITATIONS: Decreased ability to generalize the data to breast cancer survivors due to small sample size from one institution and potential referral bias. CONCLUSIONS: Cancer survivors experience QOL issues throughout the continuum of their care, which can result in long-term effects on their physical, functional, social and emotional well-being. QOL is a major focus for cancer survivors and many times determines a survivor's healthcare decisions. QOL measurements can be utilized at multiple points during survivorship to identify the need for referrals and to guide interventions.

Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein.

Geminiviruses pose serious threat to many economically important crops such as mungbean, tomato, cotton, etc. To devise a specific antiviral strategy at the viral DNA replication level, a hammerhead ribozyme was directed against the mRNA of the replication initiator protein (Rep). Rep is the most important viral protein for the DNA replication of the Mungbean yellow mosaic India virus (MYMIV), a member of the Geminiviridae family. The ribozyme showed approximately 33% cleavage activity on synthetic rep transcript within 1h under in vitro conditions, whereas the mutant ribozyme, designed to lack the catalytic activity but target the same site, showed no cleavage. The in vivo efficiency of ribozyme was evaluated in Saccharomyces cerevisiae as it can act as a surrogate host for replication of the MYMIV-DNA and lacks RNAi machinery. In the presence of the ribozyme, growth of the yeast cells that are dependent on geminiviral replication was inhibited by 30% and cellular generation time was increased by 2h. The RT-PCR analysis showed a maximum of about 50% reduction in the rep mRNA level in presence of the ribozyme compared to its noncatalytic mutant control. About 65% decrease in geminiviral DNA replication was observed due to the downregulation of replication initiator protein by the ribozyme. These results raise the possibility of engineering resistance to geminiviruses employing the ribozyme approach.

Development of expression vectors for Escherichia coli based on the pCR2 replicon.

BACKGROUND: Recent developments in metabolic engineering and the need for expanded compatibility required for co-expression studies, underscore the importance of developing new plasmid vectors with properties such as stability and compatibility. RESULTS: We utilized the pCR2 replicon of Corynebacterium renale, which harbours multiple plasmids, for constructing a range of expression vectors. Different antibiotic-resistance markers were introduced and the vectors were found to be 100% stable over a large number of generations in the absence of selection pressure. Compatibility of this plasmid was studied with different Escherichia coli plasmid replicons viz. pMB1 and p15A. It was observed that pCR2 was able to coexist with these E. coli plasmids for 60 generations in the absence of selection pressure. Soluble intracellular production was checked by expressing GFP under the lac promoter in an expression plasmid pCR2GFP. Also high level production of human IFNgamma was obtained by cloning the h-IFNgamma under a T7 promoter in the expression plasmid pCR2-IFNgamma and using a dual plasmid heat shock system for expression. Repeated sub-culturing in the absence of selection pressure for six days did not lead to any fall in the production levels post induction, for both GFP and h-IFNgamma, demonstrating that pCR2 is a useful plasmid in terms of stability and compatibility. CONCLUSION: We have constructed a series of expression vectors based on the pCR2 replicon and demonstrated its high stability and sustained expression capacity, in the absence of selection pressure which will make it an efficient tool for metabolic engineering and co-expression studies, as well as for scale up of expression.

Molecular targets for design of novel inhibitors to circumvent aminoglycoside resistance.

Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and Gram-negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Antibiotic resistance is a growing problem for all classes of anti-infective agents. One of the first groups of antibiotics to encounter the challenge of resistance was the aminoglycoside -aminocyclitol family. Initially, the resistance that emerged in organisms such as Mycobacterium tuberculosis was restricted to modification of the antibiotic targets, which we now know to be the bacterial ribosomal rRNA and proteins. As new aminoglycosides came to the clinic, however, the prevalence of chemical modification mechanisms of resistance became dominant. Enzymatic modification of aminoglycosides through kinases (O-phosphotransferases, APHs), O-adenyltransferases (ANTs) and N-acetyltransferases (AACs) has emerged in virtually all clinically relevant bacteria of both Gram-positive and Gram-negative origin. Although their clinical use has been extensive, their toxicity and the prevalence of resistance in clinical strains have prompted the pharmaceutical industry to look for alternatives. Whereas the search for novel targets for antibiotics from the genomic information is ongoing, no antibacterial agent based on these efforts has so far entered clinical trials. Meanwhile, structural knowledge of the ribosome, the target for aminoglycosides, has invigorated the field of antibiotic development. It is expected that knowledge of the binding interactions of aminoglycosides and the ribosome would lead to concepts in drug design that would take us away from the parental structures of aminoglycosides in the direction of different structural classes that bind to the same ribosomal target sites as aminoglycosides. The challenge to ensure the continued use of these highly potent antibacterial agents will require the effective management of resistance at several levels. One potential mechanism of circumventing resistance is the development of inhibitors of modification enzymes, a methodology that is now well established in the beta-lactam field. This approach requires knowledge of resistance at the molecular and atomic levels for the rational design of inhibitory molecules. The understanding of the molecular basis for aminoglycoside resistance modification has been greatly enhanced by the recent availability of representative 3D-structures from the three classes of modifying enzymes: kinases, acetyltransferases and adenyltransferases. The challenge is now to firmly establish the mechanisms of enzyme action and to use this information to prepare effective and potent inhibitors that will reverse antibiotic resistance. In this review, we discuss the molecular mechanisms of resistance of aminoglycosides specifically on aminoglycoside-modifying enzymes and newly developed strategies to circumvent resistance including antisense technology, which is an example of new strategy to deal with antibiotic resistance.

Plasmids of corynebacteria.

Corynebacteria are pleomorphic, asporogenous, Gram-positive bacteria. Included in this group are nonpathogenic soil corynebacteria, which are widely used for the industrial production of amino acids and detergents, and in biotransformation of steroids. Other members of this group are plant and animal pathogens. This review summarizes the current information available about the plasmids of corynebacteria. The emphasis is mainly on the small plasmids, which have been used for construction of vectors for expression of genes in these bacteria. Moreover, considerable information is now available on their nucleotide sequence, gene organization and modes of replication, which would make it possible to further manipulate these plasmids. Other plasmid properties, such as incompatibility and host range, are also discussed. Finally, use of these plasmids as cloning vectors for the expression of heterologous proteins using corynebacteria as hosts is also summarized to highlight the potential of these bacteria as hosts for recombinant DNA.

Construction of fusion vectors of corynebacteria: expression of glutathione-S-transferase fusion protein in Corynebacterium acetoacidophilum ATCC 21476.

A series of fusion vectors containing glutathione-S-transferase (GST) were constructed by inserting GST fusion cassette of Escherichia coli vectors pGEX4T-1, -2 and -3 in corynebacterial vector pBK2. Efficient expression of GST driven by inducible tac promoter of E. coli was observed in Corynebacterium acetoacidophilum. Fusion of enhanced green fluorescent protein (EGFP) and streptokinase genes in this vector resulted in the synthesis of both the fusion proteins. The ability of this recombinant organism to produce several-fold more of the product in the extracellular medium than in the intracellular space would make this system quite attractive as far as the downstream processing of the product is concerned.

Intergeneric protoplast fusion between xylanase producing Bacillus subtilis LYT and Corynebacterium acetoacidophilum ATCC 21476.

Hybrids between a strain of Bacillus subtilis isolated in our laboratory and having the ability to degrade xylan and other complex polysaccharides and Corynebacterium acetoacidophilum, a lysine producer, were prepared by protoplast fusion. Based on distinctive parental biochemical characteristics the fusants were grouped into 9 categories, viz. BC1 through BC9. Three of the hybrids, BC5, BC7a and BC7b, were tested for their ability to produce xylanase and lysine. Both BC7a and BC7b produced xylanase but BC5 did not, however all of them produced lysine albeit to different degrees. These results demonstrate that intergeneric gene transfer takes place through protoplast fusion between these 2 important genera of bacteria and some of the fusants inherit the useful traits of both the parents.

Evaluation of methods for the determination of Q and K antigens of an 02:K1(L) strain of Escherichia coli.

Tests made on ten colonies from a strain of Escherichia coli O2:K1 demonstrated that bacterial agglutination tests were reliable for identifying the O antigen of serogroup O2 but were unreliable for identifying the K1 antigen. The granular nature of K agglutination was not a reliable characteristic of the L type of K antigen. In contrast, indirect haemagglutination, immunodiffusion and immunoelectrophoresis tests with bacterial extracts gave consistent results with all colonies. The polysaccharide K1 antigen formed a long anodic precipitation line with two peaks, indicating its heterogenous nature, and partial fusion of this line with the O-antigen precipitation line suggested the presence of common serological determinants. In addition, a heat-labile protein antigen, possibly another K antigen, was identified by indirect haemagglutination tests and may have produced a short anodic precipitation line. The results also showed that the K1 antigen was still produced after storage of a culture for 12 years on Dorset-egg medium.

Construction of vector of Brevibacterium lactofermentum and study of its stability in continuous culture.

A 5.7-kb vector plasmid pBK2 was constructed by ligating the kanamycin resistance gene from Escherichia coli plasmid pACYC177 to an endogenous cryptic 4.4-kb plasmid of Brevibacterium lactofermentum ATCC 21086. The vector replicates efficiently and is stably maintained in the host and other coryneforms. However, the copy number varied from 50 to 10 per chromosome-equivalent under different culture conditions. Continuous culture studies showed instability when low dilution rates were used. Co-culture experiments were performed at various dilution rates to measure the growth rate ratio (alpha) of the plasmid-free cells to the plasmid-containing cells. It was observed that at low dilution rates the value of alpha was higher than that at high dilution rates. Thus, the instability of the plasmid can be attributed to the increase in alpha at low dilution rates. Modelling of instability using a random partitioning model of plasmid segregation and experimentally obtained values of alpha showed agreement with experimental data. This demonstrated that active partitioning is not the operative mechanism for plasmid segregation in this case.

Five-year follow up of the ATS mechanical heart valve.

BACKGROUND AND AIM OF THE STUDY: Between January 1, 1997 and December 31, 2001, a total of 342 patients underwent aortic valve replacement (AVR) or mitral valve replacement (MVR) with the ATS Medical prosthesis. The initial three-year phase of this study took place under a United States Food and Drug Administration-approved investigational device exemption study. The study aim was to determine the incidence of valve-related events in up to five years of follow up after valve implantation, and to assess patient disturbance from valve noise. METHODS: Patients were consecutively enrolled to undergo AVR or MVR with the ATS prosthesis. Follow up studies were conducted by patient questionnaire and/or telephone call. Follow up was 96% complete. AVR was conducted in 246 patients (80 with coronary bypass), and MVR in 96 patients (29 with coronary bypass). RESULTS: The overall operative mortality was 2.6% (n = 9; AVR 3.2%, n = 8; MVR 1.0%, n = 1), with two deaths being valve-related (0.6%). In 878 patient-years (pt-yr) of follow up (613 pt-yr for AVR; 265 pt-yr for MVR) there were an additional 22 deaths. Five deaths (0.6%/pt-yr) were valve-related: two were neuroembolic (both MVR), one from endocarditis (AVR), and two from bleeding events (both AVR). Late valve-related complications (>30 days) included 17 episodes of major bleeding (11 AVR, 1.8%/pt-yr; six MVR, 2.3%/pt-yr), five permanent neuroembolic events (four AVR, 0.7%/pt-yr; one MVR, 0.4%/pt-yr); 16 transient neuroembolic events (10 AVR, 1.6%/pt-yr; six MVR, 2.3%/pt-yr); three transient peripheral emboli (two AVR, 0.3%/pt-yr; one MVR, 0.4%/pt-yr); four paravalvular leaks (two AVR, 0.3%/pt-yr; two MVR, 0.8%/pt-yr); and one episode of valve thrombosis (MVR, 0.4%/pt-yr; AVR, 0%/pt-yr). Reoperation was required in two patients: one AVR (paravalvular leak, 0.2%/pt-yr) and one MVR (replacement due to thrombosis, 0.4%/pt-yr). CONCLUSION: These results indicate that intermediate-term results with the ATS mechanical prosthesis continue to be excellent, though further long-term follow up is warranted.

Laboratory trials with inactivated vaccines against Escherichia coli (O2:K1) infection in fowls.

Laboratory trials were carried out with an O2:K1 vaccine prepared with either the Freund's complete or incomplete adjuvant. Both types of vaccine administered subcutaneously were highly effective against a challenge with the vaccine strain within three to four weeks after vaccination at two to three weeks of age. The complete adjuvant vaccine was more effective than the incomplete adjuvant vaccine when administered to chickens of an earlier age, and in the rate of development and duration of immunity. The efficacy of both vaccines was unimpaired by their incorporation with the Newcastle disease oil adjuvant (inactivated) vaccine (Newcadin). The use of an oil adjuvant vaccine was not found to affect the rate of growth adversely or to produce any other reaction prejudicial to its commerical application. The efficacy of the vaccines was unimpaired by their incorporation with Newcastle disease oil adjuvant (inactivated) vaccine (Newcadin) thus demonstrating the possibility of producing a combined Escherichia coli/Newcastle disease virus vaccine.