title: Adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine.

text: A virulent bovine coronavirus isolate (newborn calf diarrheal) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). The same cells have been used to produce modified live porcine rotavirus and coronavirus vaccines that are federally licensed and sold worldwide. Growth of the bovine coronavirus resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Virus yields were relatively high in the ST cells and active replication was confirmed by immune electron microscopy and immunofluorescence. Adaptation of bovine coronavirus to a diploid swine cell line has not been previously reported. Different cell culture passage levels of bovine coronavirus were evaluated by oral inoculation of clean-catch, colostrum-deprived calves. A passage level of bovine coronavirus was identified that multiplied in the calf without the clinical signs of disease associated with virulent passages. The modified live bovine coronavirus vaccine remained safe and efficacious even after 5-backpassages in calves. Further efficacy studies have shown that the modified live bovine coronavirus vaccine significantly protected calves from highly virulent challenges with either winter dysentery or newborn calf diarrheal coronavirus isolates.


title: Transoral endoluminal approach to Zenker's diverticulum using Ligasure™. Early clinical experience.

text: The development of minimally invasive procedures has rekindled interest in endoluminal techniques for the management of Zenker's diverticulum. Tissue sealers as employed in laparoscopic surgery have not been previously used for Zenker's diverticulum septotomy.Supported by the established safety of linear cutters, bipolar forceps, and ultrasonic scalpels, we have started a procedure using the Ligasure 5™ tissue sealer. Safety and efficacy results in our early clinical experience are shown for a prospective series of 5 consecutive Zenker's diverticulum cases that were perorally managed with tissue sealing.The procedure was quickly and safely performed in the endoscopy room under sedation. Mean number of seals per patient was 2, and mean procedure duration was 33 minutes. No complications developed during or after the procedure, and patients were discharged with immediate dysphagia relief and adequate oral tolerance. No diverticular relapses occurred after a mean follow-up of 21 months (range 18-30). This procedure may be repeated as often as desired with no need for hospital admission. Safety should be prospectively assessed by further studies using a higher number of procedures.


[Preparing for the next pandemic].


title: Evaluation of a newly designed flow diverter for the treatment of intracranial aneurysms in an elastase-induced aneurysm model, in New Zealand white rabbits.

text: INTRODUCTION In this study, we analyzed angiographic and histologic aneurysm occlusion of a newly designed flow-diverting device. Visibility and flexibility, as well as occlusions of side branches and neointimal proliferation were also evaluated. METHODS Aneurysms were induced in 18 New Zealand white rabbits and treated with a braided, "closed-loop-designed" device of nitinol. Additional devices were implanted in the abdominal aorta to cover the origin of branch arteries. Angiographic follow-ups were performed immediately after placement of the device, after 3 months (n = 9) and 6 months (n = 9). The status of aneurysm occlusion (using a five-point scale) and the patency of branch arteries were assessed. RESULTS Aneurysm occlusion rates were noted as grade 0 in 2 (11 %), grade I in 1 (6 %), grade II in 1 (6 %), grade III in 9 (50 %), and grade IV in 5 (28 %) of 18 aneurysms, respectively, indicating a complete or near-complete occlusion of 78 % under double antiplatelet therapy. Aneurysm occlusion was significantly higher at 6 months follow-up (P = 0.025). Radiopaque markers provided excellent visibility. Limited device flexibility led to incomplete aneurysm neck coverage and grade 0 occlusion rates in two cases. Distal device occlusions were found in three cases, most likely due to an extremely undersized vessel diameter in the subclavian artery. No case of branch artery occlusion was seen. Intimal proliferation and diameter stenosis were moderate. CONCLUSION The tested flow diverter achieved near-complete and complete aneurysm occlusion under double antiplatelet therapy of elastase-induced aneurysms in 78 %, while preserving branch arteries.


title: Antiviral strategies against human coronaviruses.

text: Since the mid 60's the human coronaviruses (HCoV), represented by HCoV-OC43 and HCoV-229E, were generally considered relatively harmless viruses. This status changed dramatically with the emergence of SARS-CoV in 2002/2003. The SARS-CoV pandemic took 774 lives around the globe and infected more than 8000 people in 29 countries. SARS-CoV is believed to be of zoonotic origin, transmitted from its natural reservoir in bats through several animal species (e.g., civet cats, raccoon dogs sold for human consumption in markets in southern China). The epidemic was halted in 2003 by a highly effective global public health response, and SARS-CoV is currently not circulating in humans. The outbreak of SARS-CoV and the danger of its re-introduction into the human population, as well as the danger of the emergence of other zoonotic coronaviral infections triggered an intense survey for an efficient treatment that resulted in the evaluation of several anticoronaviral compounds. HCoV-NL63 and HCoV-HKU1 were identified shortly after the SARS-CoV outbreak. The 4 human coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 cause mild respiratory illnesses when compared to SARS, but these infections are involved in 10 - 20 % of hospitalizations of young children and immunocompromised adults with respiratory tract illness. Therefore, there is an urgent need for a successful therapy to prevent disease induction or a vaccine to prevent new infections. This review summarizes the current status of anticoronaviral strategies.


15 December News.


title: Acute or chronic immunoneutralization of somatostatin does not affect growth hormone or thyroid hormone secretion in sheep.

text: The effect of acute or chronic immunoneutralization of somatostatin (SRIF) on plasma GH, thyrotrophin (TSH) and thyroid hormones was examined. Acute responses to SRIF immunoneutralization were examined using 30 intact male lambs (19.8 +/- 0.6 kg) assigned to one of five treatment groups such that control (C) lambs received no anti-SRIF immunoglobulin and SRIF-immunized (SI) lambs received 2 mg (SI2), 10 mg (SI10), 20 mg (SI20) or 100 mg (SI100) anti-SRIF immunoglobulin/kg body weight (BW). Control immunoglobulin was administered such that all lambs received 100 mg total immunoglobulin protein/kg BW. Effects of chronic SRIF immunoneutralization were examined using C and SI100 lambs which received additional (40 mg/kg BW) control and anti-SRIF immunoglobulin respectively, 4 and 8 days following the initial dose. Blood samples were collected from all lambs, at 10-min intervals, for 5 h immediately following initial immunoglobulin infusion and, from C and SI100 lambs, at 10-min intervals, for 5 h at 11 days following initial immunoglobulin infusion. At the end of each 5-h sampling period, pituitary and thyroid function was examined by i.v. challenge with thyrotrophin-releasing hormone (TRH; 0.33 microgram/kg BW). Basal plasma GH and thyroxine (T4) and the GH, TSH, T4 and tri-iodothyronine (T3) responses to TRH were not influenced by acute or chronic immunoneutralization of SRIF. Acute, but not chronic, immunoneutralization of SRIF elevated basal plasma T3 in SI100 lambs only. The results suggest that SRIF, under physiological conditions, does not influence GH or thyroid hormone secretion in sheep but may influence thyroid hormone metabolism acutely.


Exchanging health lessons globally.


title: Cutting Edge: TLR2 is required for the innate response to Porphyromonas gingivalis: activation leads to bacterial persistence and TLR2 deficiency attenuates induced alveolar bone resorption.

text: Periodontitis is a chronic inflammatory disease that leads to destruction of the attachment apparatus of the teeth. The presence of particular oral bacteria and the host inflammatory response contribute to disease progression. Porphyromonas gingivalis is a Gram-negative anaerobe considered to be a major periodontal pathogen. Isolated Ags from P. gingivalis activate innate immune cells through TLR2 or TLR4. We challenged TLR2- and TLR4-deficient mice with live P. gingivalis and studied the inflammatory response and bacterial survival. Wild-type and TLR4-deficient mice produced high levels of cytokines in response to P. gingivalis challenge, whereas cytokine levels were nearly absent or delayed in TLR2-deficient mice. Surprisingly, P. gingivalis was cleared far more rapidly in TLR2-deficient mice. In addition, TLR2-deficient mice resisted bone loss following oral infection with P. gingivalis.


RNA sequence analysis of the E2 genes of wildtype and neuroattenuated mutants of MHV-4 reveals a hypervariable domain.


title: [Changes in pulmonary function in SARS patients during the three-year convalescent period].

text: OBJECTIVE To investigate the patterns of pulmonary function in severe acute respiratory syndrome (SARS) patients during three-year convalescent period, and to investigate the changes and the medium and long term effects on pulmonary function of SARS patients. METHODS Pulmonary function tests were conducted for four times in 37 SARS patients during three-year convalescent period. They were discharged from hospital within one month, three months, one year and three years respectively. At the same time, pulmonary function of 15 healthy persons was examined as controls to be used for comparison. RESULTS Compared with the healthy controls, there were significant decrease in forced vital capacity (FVC), vital capacity (VC), one second forced expiratory volume (FEV1), 25%-75% forced expiratory flow (25%-75%FEF) of SARS patients within three years after their discharge from hospital (all P<0.05). There were no significant differences in FEV1/FVC, total lung capacity (TLC), diffusing capacity of the lung for carbon monoxide (DLCO) between the two groups (all P>0.05). Abnormality rate of DLCO in the SARS patients within three years after discharge was significantly decreased (32.4% vs. 5.4%,P<0.05), but no significant differences in FVC, VC, 25%-75%FEF among SARS patients discharged one month and three years from the hospital (all P>0.05). Ten SARS patients showed lower FVC (mild degree in 9 cases, severe degree in 1 case) three years later, 2 patients showed mildly lowered DLCO. CONCLUSION The lung diffusion function of the SARS patients had recovered after they were discharged from hospital within three years, but in 20%-30% patients there is still mild or moderate restrictive ventilation function abnormality and small airway function impairment. The lung functions of most patients have recovered gradually, but in a minority of patients they may be impaired.


Prevention and treatment of swine-origin influenza virus with interferon: an in vivo and ex vivo study.


SARS-associated coronavirus.


title: Endocrine hypertension: An overview on the current etiopathogenesis and management options.

text: Endocrine causes of secondary hypertension include primary aldosteronism, pheochromocytoma, cushing's syndrome, hyperparathyroidism and hypo- and hyperthyroidism. They comprise of the 5%-10% of the causes of secondary hypertension. Primary hyperaldosteronism, the most common of the endocrine cause of hypertension often presents with resistant or difficult to control hypertension associated with either normo-or hypokalemia. Pheochromocytoma, the great mimicker of many conditions, is associated with high morbidity and mortality if left untreated. A complete history including pertinent family history, physical examination along with a high index of suspicion with focused biochemical and radiological evaluation is important to diagnose and effectively treat these conditions. The cost effective targeted genetic screening for current known mutations associated with pheochromocytoma are important for early diagnosis and management in family members. The current review focuses on the most recent evidence regarding causes, clinical features, methods of diagnosis, and management of these conditions. A multidisciplinary approach involving internists, endocrinologists and surgeons is recommended in optimal management of these conditions.


title: Whole Genome Analysis and Targeted Drug Discovery Using Computational Methods and High Throughput Screening Tools for Emerged Novel Coronavirus (2019-nCoV).

text: A novel coronavirus designated as SARS-CoV-2 in February 2020 by World Health organization (WHO) was identified as main cause of SARS like pneumonia cases in Wuhan city in Hubei Province of China at the end of 2019. This been recently declared as Global Pandemic by WHO. There is a global emergency to identify potential drugs to treat the SARS-CoV-2. Currently, there is no specific treatment against the new virus. There is a urgency to identifying potential antiviral agents to combat the disease is urgently needed. An effective and quick approach is to test existing antiviral drugs against. Whole genome analysis and alignment carried out using BLASTn, SMART BLAST and WebDSV 2.0 had shown more than 238 ORF's coding for proteins mostly origin from Bat SARS coronavirus and root genomic origin from Archaea. Molecular docking results against protein targets Furin, papain like proteases, RdRp and Spike glycoprotein had shown paritaprevir, ritonavir, entecavir and chloroquine derivatives are the best drugs to inhibit multi targets of coronavirus infection including natural compounds corosolic acid, baicalin and glycyrrhizic acid with minimal inhibitory concentrations. Thus we propose use of paritaprevir, entecavir, ritonavir and chloroquine derivatives as best drug combination along with niacinamide, folic acid and zinc supplements to treat novel coronavirus infection. We also propose use of plant protease inhibitors (PI's) and Anti-IL8, IL-6, IL-2 as future drug models against coronavirus.


title: Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014.

text: Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral RNA was detected in milk of five of these seven camels. The presence of MERS-CoV RNA in milk of camels actively shedding the virus warrants measures to prevent putative food-borne transmission of MERS-CoV.


title: Pathogen subversion of cell-intrinsic innate immunity.

text: The mammalian immune system has evolved under continuous selective pressure from a wide range of microorganisms that colonize and replicate in animal hosts. A complex set of signaling networks initiate both innate and adaptive immunity in response to the diverse pathogens that mammalian hosts encounter. In response, viral and microbial pathogens have developed or acquired sophisticated mechanisms to avoid, counteract and subvert sensors, signaling networks and a range of effector functions that constitute the host immune response. This balance of host response and pathogen countermeasures contributes to chronic infection in highly adapted pathogens that have coevolved with their host. In this review we outline some of the themes that are beginning to emerge in the mechanisms by which pathogens subvert the early innate immune response.


title: Regulation of coronavirus RNA transcription is likely mediated by protein-RNA interactions.

text: Coronavirus mRNA transcription was thought to be regulated by the interaction between the leader RNA and the intergenic (IG) sequence, probably involving direct RNA-RNA interactions between complementary sequences. In this study, we found that a 9-nucleotide sequence immediately downstream of the leader RNA up-regulated mRNA transcription and that a particular strain of mouse hepatitis virus (MHV) lacking this 9-nucleotide transcribed subgenomic mRNA species containing unusually heterogeneous leader-fusion sites. These results suggest that the sequence complementarity between the leader and IG is not necessarily required for mRNA transcription. UV cross-linking experiments using cytoplasmic extracts of uninfected cells and the IG sequence showed that three different cellular proteins bound to IG of the template RNA. Deletion analyses and site-directed mutagenesis of IG further demonstrated a correlation between protein-binding and transcription efficiency, suggesting that these RNA-binding proteins are involved in the regulation of coronavirus mRNA transcription. We propose that coronavirus transcription is regulated by RNA-protein and protein-protein interactions.


title: Seco-pregnane steroids target the subgenomic RNA of alphavirus-like RNA viruses.

text: Plants have evolved multiple mechanisms to selectively suppress pathogens by production of secondary metabolites with antimicrobial activities. Therefore, direct selections for antiviral compounds from plants can be used to identify new agents with potent antiviral activity but not toxic to hosts. Here, we provide evidence that a class of compounds, seco-pregnane steroid glaucogenin C and its monosugar-glycoside cynatratoside A of Strobilanthes cusia and three new pantasugar-glycosides of glaucogenin C of Cynanchum paniculatum, are effective and selective inhibitors to alphavirus-like positive-strand RNA viruses including plant-infecting tobacco mosaic virus (TMV) and animal-infecting Sindbis virus (SINV), eastern equine encephalitis virus, and Getah virus, but not to other RNA or DNA viruses, yet they were not toxic to host cells. In vivo administration of the compounds protected BALB/c mice from lethal SINV infection without adverse effects on the mice. Using TMV and SINV as models, studies on the action mechanism revealed that the compounds predominantly suppress the expression of viral subgenomic RNA(s) without affecting the accumulation of viral genomic RNA. Our work suggested that the viral subgenomic RNA could be a new target for the discovery of antiviral drugs, and that seco-pregnane steroid and its four glycosides found in the two medicinal herbs have the potential for further development as antiviral agents against alphavirus-like positive-strand RNA viruses.


title: Applying a cultural lens to the concept of burnout.

text: Burnout is a unique type of stress syndrome that is pervasively affecting the nursing profession. The literature on burnout is reviewed, and a conceptual framework using Maslach's Burnout Inventory is provided. The culture of organizations is examined with regard to its contribution to the antecedents and consequences of burnout. Leininger's cultural care theory and its relevance to the concept of burnout and culture is discussed. Implications for the amelioration of burnout from the perspective of culture are discussed to stimulate future changes in nursing administration, practice, and education. A culture of trust, support, open communication, and respect can foster healthy work environments and protect against burnout.


title: Cryptosporidium and host resistance: historical perspective and some novel approaches.

text: Cryptosporidium parvum is recognized as a major cause of diarrheal disease in neonatal bovine calves. In addition, this protozoan parasite has emerged as an important cause of disease in both immunocompromised and immunocompetent humans. Despite years of research, no consistently effective means of prevention or treatment are readily available for cryptosporidiosis in any species. Infection through ingestion of contaminated water has been widely documented; C. parvum was reported to be responsible for the largest waterborne outbreak of infectious disease in US history. In addition to its role as a primary disease agent, C. parvum has potential to initiate or exacerbate other gastrointestinal disorders, such as inflammatory bowel disease. Thus, control of C. parvum infection in both animals and humans remains an important objective. Research in our laboratory has focused on understanding mechanisms of resistance to C. parvum. We have demonstrated that acquisition of intestinal flora increases resistance to C. parvum. Substances present in the intestinal mucosa of adult animals can transfer resistance when fed to susceptible infants. Both expression of intestinal enzymes and rate of proliferation of epithelial cells may be altered following C. parvum infection. These and other changes may have profound effects on host resistance to C. parvum.


title: Olfactory neuropathy in severe acute respiratory syndrome: report of A case.

text: This case was a 27 years old female with severe acute respiratory syndrome (SARS). She suffered from typical symptoms of SARS. Although she got almost complete recovery from most symptoms after treatment, she noted acute onset complete anosmia 3 weeks after the onset of her first symptom. Her brain MRI examination did not show definite lesion except an incidental finding of left temporal epidermoid cyst. Her anosmia persisted for more than 2 years during following up. Peripheral neuropathy and myopathy have been reported as a concomitant problem during the convalescent stage of SARS, while the sequel of permanent ansomia in SARS was not reported before. Olfactory neuropathy, which rarely occurred in typical peripheral neuropathy, could be a special type of neuropathy induced by corona virus infection in SARS. Olfactory function test should be taken into routine check-up for patients with SARS. The pathophysiology and therapeutic strategy of this special type of permanent olfactory dysfunction deserve further investigation.


title: [COVID-19 - A RECENT REVIEW].

text: INTRODUCTION Corona disease sprang into our lives towards the end of 2019. In November/December 2019, in Wuhan, Hubei province in China, a SARS-like viral disease with a high adhesive capacity and the potential to endanger the life of the patient was reported. It soon became clear that the disease was very contagious and new hospitals were being built on the site and a General closure was underway. Many doctors from all over China were recruited and sent to the isolated area. Local medical reports were acute respiratory distress syndrome (ARDS)-like, and patients needed respiratory support for oxygenation and some needed ventilation on respiratory devices with significant mortality rates. The disease has since spread to many parts of the world, mainly towards Europe. Hospitals have received hundreds of patients, to the point of partially collapsing the glorious Italian healthcare system. Italy and Spain reported a lung disease that causes severe oxidative disruption but no disruption in ventilation unlike ARDS. Initial post-mortem shows that the main cause of death is thromboembolic. Heart involvement with myocarditis and even heart attacks are beginning to be reported. Various drugs are involved in the treatment plan and are often known to fail. Even promising remedies such as Remdesavir, which is an antiviral drug, are beginning to receive reports from controlled trials, which are not as successful as originally expected. All in all, we continue to wander in the dark, both about the disease and certainly, regarding the treatment. The primary treatment involves maximum efforts for respiratory, hematologic, nephrologic and cardiologic support. In this review, I will try to describe the disease and the enigma concerning suggested treatments.


title: Severe, diffuse fibrinonecrotic pleuropneumonia in a cat affected by multiple viral infection.

text: This communication describes the coinfection with feline panleukopenia virus (FPV), feline herpesvirus 1 (FeHV-1), feline calicivirus (FCV) and feline coronavirus (FCoV) in a 1 year‑old domestic cat living in a feline shelter. The cat was referred to veterinary hospital with clinical signs related to diffuse gastro-intestinal inflammation, it had developed a severe pneumopathy with fibrinous exudation in all body cavities and died 8 days after initial presentation. Pathological findings and biomolecular diagnostic test results were compatible with an initial FPV infection that, in consequence of the lymphoid depletion, has fostered coinfection or reactivation of chronic-latent infections with FeHV-1, FCV, and FCoV. In the reported case, the simultaneous presence of different viruses exacerbated the clinical status of the host, resulting in multiple organ damage and leading it to its death.


title: Application of a snare technique in retrograde chronic total occlusion percutaneous coronary intervention - a step by step practical approach and an observational study.

text: Percutaneous coronary intervention (PCI) for chronic total occlusion (CTO) has recently become popular among interventional cardiologists. CTO originating from the ostium has been one of the most difficult CTO lesions to treat with PCI for a number of reasons. Our aim was to illustrate a specific technique during retrograde CTO PCI referred to as the "snare technique."We retrospectively examined the use of "snare technique" among 371 consecutive retrograde CTO PCIs performed at our institution between 2006 and 2015."Snare technique" was used in 10 patients among the 371 retrograde CTO PCIs. The baseline clinical and angiographic characteristics of patients with or without "snare technique" were similar. The "snare technique" group had significantly fewer side branches at occlusion (30.0% vs 71.2%, P = 0.01) and a higher incidence of externalization (90% vs 25.5%, P < 0.001). The contrast volume was significantly lower in the "snare technique" group (285.0 ± 68.5 vs 379.2 ± 144.0, P = 0.04). The incidence of major complications, retrograde success, or final success did not differ between the groups.The "snare technique" is safe and feasible in retrograde CTO PCI, especially in cases of difficult coronary engagement in cases such as ostial occlusion, challenging coronary anatomy, or retrograde guidewire cannot get in antegrade guiding catheter.


Apocalypse or redemption: responding to extensively drug-resistant tuberculosis.


title: Impact of robotic partial nephrectomy with and without ischemia on renal functions: experience in 34 cases.

text: OBJECTIVE In this study we aimed to compare renal functions in patients who underwent robotic partial nephrectomy (RPN) with on-clamp and zero- ischemia techniques. MATERIAL AND METHODS Between 2009 and 2015, 12 off-clamp and 22 on-clamp RPN procedures were performed on a total of 34 patients in two centers. The main outcome parameters examined were serum creatinine, and estimated glomerular filtration rate (eGFR) during preoperative, immediate postoperative periods, and at postoperative 3rd months. RESULTS There were no statistically significant differences between on-clamp and zero- ischemia groups regarding age, ASA score, BMI, PADUA and R.E.N.A.L. nephrometry scores, operation time and tumor size (p>0.05). Significant differences were found in the duration of hospital stay (3.8±0.9 days vs. 3.0±0.9 days) and amount of blood loss (85.9±49.6 mL vs. 183.3±176.2 mL) between the on-clamp and zero-ischemia groups (p<0.05). Statistically significant differences were found between preoperative and immediate post-operative periods, in terms of eGFR and serum creatinine levels in both groups. Moreover, statistically significant differences were found between preoperative and postoperative 3rd month periods, in the on-clamp group in terms of eGFR and serum creatinine levels. In the zero-ischemia group, the decrease in eGFR and serum creatinine levels at postoperative 3rd month relative to the preoperative period was not statistically significant. CONCLUSION Off-clamp RPN technique is superior, in short-term outcomes involving renal functions, compared to on clamp approach. However, long- term data regarding the renal functions should be evaluated to arrive at a definitive decision.


title: Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1.

text: Intranasal inhalation of the severe acute respiratory syndrome coronavirus (SARS CoV) in the immunocompetent mouse strain 129SvEv resulted in infection of conducting airway epithelial cells followed by rapid clearance of virus from the lungs and the development of self-limited bronchiolitis. Animals resistant to the effects of interferons by virtue of a deficiency in Stat1 demonstrated a markedly different course following intranasal inhalation of SARS CoV, one characterized by replication of virus in lungs and progressively worsening pulmonary disease with inflammation of small airways and alveoli and systemic spread of the virus to livers and spleens.


title: Rarity gives a charm: evaluation of trace proteins in plasma and serum.

text: Since plasma potentially contacts every cell as it circulates through the body, it may carry clues both to diagnosis and treatment of disease. It is commonly expected that the growing ability to detect and characterize trace proteins will result in discovery of novel therapeutics and biomarkers; however, the familiar, super-abundant plasma proteins remain a fundamental stumbling block. Furthermore, robust validation of proteomic data is a sometimes overlooked but always necessary component for the eventual development of clinical reagents. This review surveys some of the uses of typical and atypical low-abundance proteins, current analytical methods, existing impediments to discovery, and some innovations that are overcoming the challenges to evaluation of trace proteins in plasma and serum.


title: Experimental inoculation of cats with canine coronavirus and subsequent challenge with feline infectious peritonitis virus.

text: Oronasal inoculation of minimal-disease cats with a virulent field isolate of canine coronavirus elicited a neutralizing antibody response that increased steadily over a two-month period, indicating in vivo amplification of the inoculated dose. A heterologous neutralizing antibody response to transmissible gastroenteritis virus could be detected three weeks after titers to canine coronavirus were first observed and was found to parallel the homologous response at a level approximately one log10 dilution lower. A modest anamnestic response detectable in both assays was observed after a second exposure to canine coronavirus. Cross-reacting antibodies to transmissible gastroenteritis virus were not detected by a kinetics-based enzyme-linked immunosorbent assay (KELA), however, until after cats had received multiple daily doses of canine coronavirus (third exposure). No clinical signs attributable to canine coronavirus were seen in inoculated cats, and virus could not be isolated from fecal or rectal swabs nor identified in fecal specimens by electron microscopy. Both virus-neutralizing and KELA titers were boosted by aerosol challenge with feline infectious peritonitis virus. Antibodies produced in response to canine coronavirus did not sensitize cats to subsequent feline infectious peritonitis virus challenge, but neither did they protect cats against the challenge dose.


title: A PCR-enhanced method for determining the 5' end sequence of mRNAs.

text: A method was developed to amplify ligated cDNA copies of mRNA 5' ends before cloning and sequencing. The method facilitates the study of the 5' end sequence on many molecules of an individual mRNA species. It can also be used to study the 5' end sequence on other kinds of single-stranded RNA molecules.


title: Infectious pathogen detection arrays: viral detection in cell lines and postmortem brain tissue.

text: A unique array-based pathogen chip has been developed for the detection of viral RNA or DNA relevant to pathologies of the central nervous system. A total of 715 unique oligonucleotides (60-mer) representing approximately 100 pathogens were designed based on open reading frames (ORFs) from highly conserved and heterogenic regions within viral families. In addition, viral genes reflecting different stages of pathogen infection were also included to potentially define the stage of the viral infection. Viruses (double-stranded DNA, double- or single-stranded RNA, delta, retroid), parasites, and bacteria were included. Test samples labeled with Cy5 were examined by cohybridization with a reference RNA, labeled with Cy3, to the pathogen microarray chip. Good reproducibility of experiments was observed, based on data generated from duplicate hybridizations and duplicate spots on the microarray platform. A viral transcript detection sensitivity of 1 x 10(3) plaque-forming units (pfus) was achieved using selected cell lines and viruses. These findings suggest that the array-based platform described here is capable of detecting a broad spectrum of viruses in a single assay with relatively high sensitivity, specificity, and reproducibility. This method may be used to provide evidence of viral infection in postmortem tissue from psychiatric patients as well as a wide range of other diagnostic categories.


title: Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice.

text: Severe acute respiratory syndrome (SARS) is a life-threatening infectious disease which has been difficult to study and treat because of the lack of a readily available animal model. Intranasal infection of A/J mice with the coronavirus murine hepatitis virus strain 1 (MHV-1) produced pulmonary pathological features of SARS. All MHV-1-infected A/J mice developed progressive interstitial pneumonitis, including dense macrophage infiltrates, giant cells, and hyaline membranes, resulting in death of all animals. In contrast, other mouse strains developed only mild transitory disease. Infected A/J mice had significantly higher cytokine levels, particularly macrophage chemoattractant protein 1 (MCP-1/CCL-2), gamma interferon, and tumor necrosis factor alpha. Furthermore, FGL2/fibroleukin mRNA transcripts and protein and fibrin deposits were markedly increased in the lungs of infected A/J mice. These animals developed a less robust type I interferon response to MHV-1 infection than resistant C57BL/6J mice, and treatment with recombinant beta interferon improved survival. This study describes a potentially useful small animal model of human SARS, defines its pathogenesis, and suggests treatment strategies.


title: How infectious disease outbreaks affect community-based primary care physicians: comparing the SARS and H1N1 epidemics.

text: OBJECTIVE To compare how the infectious disease outbreaks H1N1 and severe acute respiratory syndrome (SARS) affected community-based GPs and FPs. DESIGN A mailed survey sent after the H1N1 outbreak compared with the results of similar survey completed after the SARS outbreak. SETTING Greater Toronto area in Ontario. PARTICIPANTS A total of 183 randomly selected GPs and FPs who provided office-based care. MAIN OUTCOME MEASURES The perceptions of GPs and FPs on how serious infectious disease outbreaks affected their clinical work and personal lives; their preparedness for a serious infectious disease outbreak; and the types of information they want to receive and the sources they wanted to receive information from during a serious infectious disease outbreak. The responses from this survey were compared with the responses of GPs and FPs in the greater Toronto area who completed a similar survey in 2003 after the SARS outbreak. RESULTS After the H1N1 outbreak, GPs and FPs still had substantial concerns about the effects of serious infectious disease outbreaks on the health of their family members. Physicians made changes to various office practices in order to manage and deal with patients with serious infectious diseases. They expressed concerns about the effects of an infectious disease on the provision of health care services. Also, physicians wanted to quickly receive accurate information from the provincial government and their medical associations. CONCLUSION Serious community-based infectious diseases are a personal concern for GPs and FPs, and have considerable effects on their clinical practice. Further work examining the timely flow of relevant information through different health care sectors and government agencies still needs to be undertaken.


Authors' reply.


title: Evaluation of the baculovirus-expressed S glycoprotein of transmissible gastroenteritis virus (TGEV) as antigen in a competition ELISA to differentiate porcine respiratory coronavirus from TGEV antibodies in pigs.

text: The spike (S) glycoprotein of the Miller strain of transmissible gastroenteritis virus (TGEV) was recently cloned and expressed in baculovirus. The recombinant S protein was used as the coating antigen in a competition (blocking) enzyme-linked immunosorbent assay (ELISA) in combination with monoclonal antibodies to the S protein epitope A (conserved on TGEV and porcine respiratory coronavirus [PRCV]) or epitope D (present on TGEV only) to differentiate PRCV- from TGEV-induced antibodies. One set (set A) of 125 serum samples were collected at different times after inoculation of caesarean-derived, colostrum-deprived (n = 52) and conventional young pigs (n = 73) with 1 of the 2 porcine coronaviruses or uninoculated negative controls (TGEV/PRCV/negative = 75/30/20). A second set (set B) of 63 serum samples originated from adult sows inoculated with PRCV and the recombinant TGEV S protein or with mock-protein control and then exposed to virulent TGEV after challenge of their litters. Sera from set A were used to assess the accuracy indicators (sensitivity, specificity, accuracy) of the fixed-cell blocking ELISA, which uses swine testicular cells infected with the M6 strain of TGEV as the antigen source (ELISA 1) and the newly developed ELISA based on the recombinant S protein as antigen (ELISA 2). The sera from set B (adults) were tested for comparison. The plaque reduction virus neutralization test was used as a confirmatory test for the presence of antibodies to TGEV/PRCV in the test sera. The accuracy indicators for both ELISAs suggest that differential diagnosis can be of practical use at least 3 weeks after inoculation by testing the dual (acute/convalescent) samples from each individual in conjunction with another confirmatory (virus neutralization) antibody assay to provide valid and complete differentiation information. Moreover, whereas ELISA 1 had 10-20% false positive results to epitope D for PRCV-infected pigs (set A samples), no false-positive results to epitope D occurred using ELISA 2, indicating its greater specificity. The progression of seroresponses to the TGEV S protein epitopes A or D, as measured by the 2 ELISAs, was similar for both sets (A and B) of samples. Differentiation between TGEV and PRCV antibodies (based on seroresponses to epitope D) was consistently measured after the third week of inoculation.


title: Upper respiratory tract infections in children.

text: Respiratory tract infections are a frequent problem for children, families, and pediatricians. Most such infections are trivial and do not warrant significant concern; however, for some children and some infections, aggressive and effective therapy is important. Much of the literature discussed in this article reflects attempts to clarify and improve appropriate use of antimicrobial therapy for those children in whom it is necessary.


title: [Expert panel recommendations on therapeutic and diagnostic management of urinary incontinence and overactive bladder in women].

text: The expert panel recommendations are issued in order to increase the number of patients with urinary incontinence and overactive bladder receiving appropriate care. The expert panel recommends that urologists, gynecologists and other physicians interested in the field of incontinence should Incontinence question should be actively asked during each physician visit and if the answer is positive it should be followed by detailed questionnaire aiming at disclosing at which occasion patient is loosing urine. The next step should be urogynecological examination and cough stress test. The panel recommends urine dipstick in all women and post void residual urine measurement only in women with voiding difficulties. Other tests, such as ultrasound, cystoscopy urodynamics are not recommended during initial diagnostic procedure. The indications for referral are significant pelvic organ prolapse, haematuria, pain during micturition, recurrent incontinence and infections, suspicion of fistula. The initial management of stress urinary incontinence should include lifestyle interventions, and physiotherapy Use of pessaries is acceptable in women who are not fit or do not want surgical therapy Local estrogen therapy should only be used in women with urogenital atrophy Duloxetin is an option in the pharmacological therapy of stress incontinence, but it doesn't cure the disease. The ineffectiveness of initial procedure should be indication to surgery Alphaadrenomimetic drugs are not recommended in the therapy of urinary incontinence. The initial management of overactive bladder and urgency incontinence should include lifestyle interventions, however fluid restrictions (if fluid load is less than 3000 ml) are not recommended. The cornerstone of overactive bladder and urgency incontinence therapy remains the treatment with anticholinergic drugs. Drugs are only effective when used accordingly to the registered doses. The new generation anticholinergics are recommended over the old ones, especially in frail elderly patients and in patients with concomitant diseases, due to their better safety profile. The evaluation of anticholinergics efficacy should be performed after 2-3 months, then after 6 months.


title: Suppression subtraction hybridization (SSH) and macroarray techniques reveal differential gene expression profiles in brain of sea bream infected with nodavirus.

text: Despite of the impact that viruses have on aquatic organisms, relatively little is known on how fish fight against these infections. In this work, the brain gene expression pattern of sea bream (Sparus aurata) in response to nodavirus infection was investigated. We used the suppression subtractive hybridization (SSH) method to generate a subtracted cDNA library enriched with gene transcripts differentially expressed after 1 day post-infection. Some of the ESTs from the infected tissues fell in gene categories related to stress and immune responses. For the reverse library (ESTs expressed in controls compared with infected tissues) the most abundant transcripts were of ribosomal and mitochondrial nature. Several ESTs potentially induced by virus exposure were selected for in vivo expression studies. We observed a clear difference in expression between infected and control samples for two candidate genes, ubiquitin conjugating enzyme 7 interacting protein, which seems to play an important role in apoptosis and the interferon induced protein with helicase C domain 1 (mda-5) that contributes to apoptosis and regulates the type I IFN production, a key molecule of the antiviral innate response in most organisms.


title: Baculovirus expression of turkey coronavirus nucleocapsid protein.

text: The nucleocapsid (N) gene of turkey coronavirus (TCV) was amplified by reverse transcriptase-polymerase chain reaction, cloned, and expressed in the baculovirus expression system. A recombinant baculovirus containing the TCV N gene (rBTCV/N) was identified by polymerase chain reaction and expression of TCV N protein as determined by western immunoblot analysis. Two TCV-specific proteins, 52 and 43 kDa, were expressed by rBTCV/N; one of these proteins, p52, was comparable in size to native TCV N protein. Baculovirus-expressed N proteins were used as antigen in an indirect enzyme-linked immunosorbent assay (ELISA) for detection of TCV-specific antibodies. The ELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related avian coronavirus, but did not detect antibodies specific for other avian viruses (avian influenza, avian reovirus, avian paramyxovirus 3, avian adenovirus 1, or Newcastle disease virus). These findings indicate that baculovirus-expressed TCV N protein is a suitable source of antigen for ELISA-based detection of TCV-specific antibodies in turkeys.


title: The effect of public health measures on the 1918 influenza pandemic in U.S. cities.

text: During the 1918 influenza pandemic, the U.S., unlike Europe, put considerable effort into public health interventions. There was also more geographic variation in the autumn wave of the pandemic in the U.S. compared with Europe, with some cities seeing only a single large peak in mortality and others seeing double-peaked epidemics. Here we examine whether differences in the public health measures adopted by different cities can explain the variation in epidemic patterns and overall mortality observed. We show that city-specific per-capita excess mortality in 1918 was significantly correlated with 1917 per-capita mortality, indicating some intrinsic variation in overall mortality, perhaps related to sociodemographic factors. In the subset of 23 cities for which we had partial data on the timing of interventions, an even stronger correlation was found between excess mortality and how early in the epidemic interventions were introduced. We then fitted an epidemic model to weekly mortality in 16 cities with nearly complete intervention-timing data and estimated the impact of interventions. The model reproduced the observed epidemic patterns well. In line with theoretical arguments, we found the time-limited interventions used reduced total mortality only moderately (perhaps 10-30%), and that the impact was often very limited because of interventions being introduced too late and lifted too early. San Francisco, St. Louis, Milwaukee, and Kansas City had the most effective interventions, reducing transmission rates by up to 30-50%. Our analysis also suggests that individuals reactively reduced their contact rates in response to high levels of mortality during the pandemic.


title: [Relationship between the severity, course, fatality of severe acute respiratory syndrome patients and the timing of hospitalization].

text: OBJECTIVE To study the relationship of timing of hospitalization and the severity, course, fatality of severe acute respiratory syndrome (SARS) patients. METHOD 1291 hospital records of clinically diagnosed SARS patients with complete data gathered from "2003 Beijing SARS Clinical Database" were analyzed. RESULTS SARS cases were categorized into four groups, according to the time of hospitalization after onset of the disease: within 3 days, during day 4 to day 7, during day 8 to day 14 and after day 14. The numbers of cases for each group were 568, 496, 177 and 50 respectively. Data showed that from group 1 to 4, the prevalence rates of major symptoms on the first day of hospitalization were: (1) 9.7%, 16.5%, 23.1% and 24.0% for "feeling chest pain" (P < 0.001), (2) 7.4%, 13.7%, 19.2% and 22.0% for "suffering from breathing obstruction" (P < 0.001), (3) 32.8%, 44.8%, 59.9% and 48.0%, for "coughing" (P < 0.001) and (4) 14.1%, 22.4%, 27.1% and 18.0% for "coughing up phlegm" (P = 0.0002), respectively. The rates of high respiratory frequency (>or= 24 bits/min.) were 11.1%, 15.5%, 22.8% and 25.5% (P < 0.001). The rates of abnormal chest X-ray were 80.3%, 89.0%, 92.3% and 88.9%, respectively (P = 0.002). The average numbers of abnormal lung field (the lung were divided into 6 fields) were 1.7, 1.9, 2.5 and 2.6 (P < 0.001); The numbers of cases receiving continuous oxygen supply treatment were 33.6%, 50.0%, 53.7% and 74.0% (P < 0.001), and the numbers of cases receiving glucocorticosteroids treatment were 28.2%, 35.9%, 53.7% and 62.0% (P < 0.001), respectively. With cases having had chronic baseline diseases prior to SARS infection, the age-standardized fatality rates were 14.9%, 11.7%, 50.0% and 33.9% (P < 0.001), and the average courses of the disease were 30.3, 34.2, 42.9 and 47.5 days (P < 0.001), respectively. In cases without chronic baseline diseases, the age-standardized fatality rates were 5.3%, 9.8%, 9.2% and 8.3% (P = 0.101), and the average courses for each group were 32.4, 35.3, 40.9 and 47.6 days (P < 0.001), respectively. CONCLUSION Delayed hospitalization would cause the situation of SARS patient to deteriorate, losing the best chance for treatment and increase case fatality. In terms of control program on SARS, emphasize should be paid on decreasing the panic of patients to the disease so as to get early hospitalization.


title: RNA of mouse hepatitis virus.

text: The RNA of mouse hepatitis virus, a coronavirus, was isolated from the virus released early in the infection and analyzed by sucrose gradient sedimentation and electrophoresis. It was found to consist of a piece of single-stranded RNA of about 60S. Its molecular weight was estimated to be 5.4 X 10(6) by electrophoresis in methylmercury-agarose gels. At least one third of the RNA contained polyadenylated sequences. It is, therefore, probably positive stranded. The virus harvested late in the infection contained, in addition to 60S, some 30 to 50S RNA that are possibly degradation products of the 60S RNA. No difference in the electrophoretic behavior could be detected between the RNA isolated from a pathogenic (JHM) and a nonpathogenic (A59) strain.


title: The Impact of SARS on Childbirth Education.

text: The SARS (Severe Acute Respiratory Syndrome) crisis in Toronto in the spring of 2003 had major consequences for prenatal learners. Classes in hospitals were cancelled; many couples were left without any prenatal education. This paper outlines the decision-making process of one programme that was determined to keep classes open. The impact of SARS on hospital procedures is also described. Childbirth educators are encouraged to prepare for future public health emergencies. Strategies to prepare for conducting childbirth education classes during times of crisis are outlined.


title: [Clinical experience from treatment of seven SARS patients].

text: OBJECTIVE To summarize the clinical experience from treatment of patients with severe acute respiratory syndrome (SARS). METHODS Retrospective analysis of seven patients with SARS in Ditan hospital treated since April 22 in 2004 was performed. RESULTS In the 7 patients, 2 were male, 5 were female, and the average age was (35.3 plus/minus 11.3) years. The main clinical manifestations were fever, cough, minor or serious dyspnea, nausea, signs of injury to other organs, and so on. The treatment regiments included oxygen, small dosage and short period of methylprednisolone (1 to 2 mg/kg), use of ventilator, psychological intervention, and treatment of underlying diseases, after which, all the 7 patients recovered. CONCLUSION Rational use of methylprednisolone and timely use of ventilator were the key steps of treatment.


title: Use of viral lysate antigen combined with recombinant protein in Western immunoblot assay as confirmatory test for serodiagnosis of severe acute respiratory syndrome.

text: A Western immunoblot assay for confirmatory serodiagnosis of severe acute respiratory syndrome (SARS) was developed utilizing viral lysate antigens combined with a recombinant nucleocapsid protein, GST-N (glutathione S-transferase-nucleocapsid) of the SARS coronavirus (SARS-CoV). The viral lysate antigens were separated by electrophoresis and transblotted onto nitrocellulose membranes. The resultant membrane was subsequently added with the GST-N recombinant protein at a specific location. The positions of bands corresponding to some of the structural proteins immobilized on the membrane were then located and verified with mouse or rabbit antisera specific to the respective proteins. The Western immunoblot assay was able to detect antibodies to SARS-CoV in all 40 serum specimens from SARS patients and differentiate the SARS-positive samples from those of the healthy donor or non-SARS patient controls (150 samples) when set criteria were followed. In addition, when the immunoblot was used to test samples considered falsely positive by an in-house-developed SARS-specific enzyme-linked immunosorbent assay, band patterns different from those with samples from SARS patients were obtained.


title: The small envelope protein E is not essential for murine coronavirus replication.

text: The importance of the small envelope (E) protein in the assembly of coronaviruses has been demonstrated in several studies. While its precise function is not clearly defined, E is a pivotal player in the morphogenesis of the virion envelope. Expression of the E protein alone results in its incorporation into vesicles that are released from cells, and the coexpression of the E protein with the membrane protein M leads to the assembly of coronavirus-like particles. We have previously generated E gene mutants of mouse hepatitis virus (MHV) that had marked defects in viral growth and produced virions that were aberrantly assembled in comparison to wild-type virions. We have now been able to obtain a viable MHV mutant in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, has been deleted. This mutant (Delta E) was obtained by a targeted RNA recombination method that makes use of a powerful host range-based selection system. The Delta E mutant produces tiny plaques with an unusual morphology compared to plaques formed by wild-type MHV. Despite its low growth rate and low infectious titer, the Delta E mutant is genetically stable, showing no detectable phenotypic changes after several passages. The properties of this mutant provide further support for the importance of E protein in MHV replication, but surprisingly, they also show that E protein is not essential.


title: The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus.

text: In addition to the RI (replicative intermediate RNA) and native RF (replicative form RNA), mouse hepatitis virus-infected cells contained six species of RNA intermediates active in transcribing subgenomic mRNA. We have named these transcriptive intermediates (TIs) and native transcriptive forms (TFs) because they are not replicating genome-sized RNA. Based on solubility in high salt solutions, approximately 70% of the replicating and transcribing structures that accumulated in infected cells by 5-6 h post-infection were multi-stranded intermediates, the RI/TIs. The other 30% were in double-stranded structures, the native RF/TFs. These replicating and transcribing structures were separated by velocity sedimentation on sucrose gradients or by gel filtration chromatography on Sepharose 2B and Sephacryl S-1000, and migrated on agarose gels during electrophoresis, according to their size. Digestion with RNase T1 at 1-10 units/microgram RNA resolved RI/TIs into RF/TF cores and left native RF/TFs intact, whereas RNase A at concentrations of 0.02 microgram/microgram RNA or higher degraded both native RF/TFs and RI/TIs. Viral RI/TIs and native RF/TFs bound to magnetic beads containing oligo(dT)(25), suggesting that the poly(A) sequence on the 3' end of the positive strands was longer than any poly(U) on the negative strands. Kinetics of incorporation of [(3)H]uridine showed that both the RI and TIs were transcriptionally active and the labelling of RI/TIs was not the dead-end product of aberrant negative-strand synthesis. Failure originally to find TIs and TF cores was probably due to overdigestion with RNase A.


title: [Optimization of expression condition of SARS-CoV PUPs genes in E. coli].

text: According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.


title: 'Getting the biosecurity architecture right' in the Asia-Pacific region.

text: This paper offers insights to enhance biosecurity in the Asia-Pacific by asking three questions. What risks/threats are significant in the region? Which security measures should be prioritized? What kind of regional--institutionalized and ad-hoc--arrangements should be implemented? By doing so the paper specifically focuses on two important aspects of biosecurity. Firstly, the issue of immediate capacity-building in the context of disaster relief, public health preparedness and biodefence. Secondly, the long-term preventive aspect, namely education and awareness-raising activities to more broadly promote responsible conduct in life science research in terms of laboratory accidents as well as the intentional use of biological agents for hostile purposes. In order to promote both aspects of biosecurity within the policy process, the paper analyses the strategic value of the US-Japan partnership in its efforts to build a biosecurity architecture in the Asia-Pacific region.


title: An investigation into respiratory tract viruses in children with acute lower respiratory tract infection or wheezing.

text: BACKGROUND This study aimed to determine the frequencies of respiratory tract viruses in patient (acute lower respiratory tract infection (LRTI) or wheezing) and control (history of asthma without symptoms) groups. METHODS Using multiplex-PCR, respiratory tract viruses were investigated in the respiratory tract specimens from patient and control groups followed in the Pediatric Clinic. RESULTS The viruses detected in the patient and control groups (p=0.013) were as follows, respectively: rhinoviruses A, B, C (25.6% and 36.7%), influenza virus A (21.1% and 0.0%), parainfluenza virus type 1 (7.8% and 1.7%), parainfluenza virus type 4 (5.6% and 0.0%), adenoviruses A, B, C, D, E (4.4% and 1.7%), parainfluenza virus type 3 (4.4% and 1.7%), coronaviruses 229E and NL63 (4.4% and 1.7%), coronavirus OC43 (3.3% and 0.0%), respiratory syncytial virus A (3.3% and 0.0%), parainfluenza virus type 2 (2.2% and 0.0%), influenza virus B (2.2% and 0.0%), and respiratory syncytial virus B (1.1% and 1.7%). No bocavirus, metapneumovirus or enterovirus was found in any specimen. Statistically significant differences in the detection of influenza virus A (p=0.000), the total detection of parainfluenza viruses (p=0.008) and co-infection (p=0.004) were observed between the patient and control groups. CONCLUSIONS The advantage of our study compared with other studies is the inclusion of not only wheezing patients but also children with asthma without symptom. The higher detection of Rhinoviruses both in patient and control groups give rise to thought that these viruses may be responsible for asthma exacerbations and may be related with long duration of virus shedding.


title: Nitric oxide up-regulates the glucocorticoid receptor and blunts the inflammatory reaction in porcine endotoxin sepsis.

text: OBJECTIVES Nitric oxide inhibits the expression of many genes involved in inflammatory diseases. Glucocorticoids inhibit similar transcription factors. We hypothesized that there may be an interaction between nitric oxide and glucocorticoids, with the potential to enhance the anti-inflammatory effect when administered simultaneously. DESIGN Prospective, randomized, controlled study. SETTING Animal research laboratory. SUBJECTS A total of 45 anesthetized and mechanically ventilated pigs. INTERVENTIONS Lung and systemic injury was induced by intravenous infusion of endotoxin (lipopolysaccharide) for 6 hrs. After 2.5 hrs, one group received 3.5 mg/kg hydrocortisone, another group inhaled nitric oxide (30 ppm), and still another group received both steroid and nitric oxide. Control groups of healthy and endotoxin-exposed piglets were also studied. MEASUREMENTS AND MAIN RESULTS Central hemodynamics and gas exchange were measured. Detection of the glucocorticoid receptor and inflammatory markers in lung, liver, and kidney tissue were made by immunohistochemistry, and morphology was studied with light microscopy. Endotoxin infusion markedly reduced glucocorticoid receptor expression in lung, liver, and kidney and up-regulated activator protein-1 and the inflammatory markers nuclear factor-kappaB and tumor necrosis factor-alpha. When administered separately, steroids and nitric oxide had modest effect on the inflammatory response. However, nitric oxide up-regulated the glucocorticoid receptor expression. Simultaneous administration of steroids and nitric oxide attenuated the inflammatory response and almost preserved or restored normal histology of both lung and systemic organs. When the glucocorticoid receptor was blocked by a receptor antagonist (mifepristone, 600 mg) and inhaled nitric oxide was subsequently administered, no increase in the expression of the glucocorticoid receptor was seen. CONCLUSION We suggest that up-regulation of glucocorticoid receptor expression by nitric oxide made steroid therapy more effective.


title: Evaluation of the effectiveness of two infectious bronchitis virus vaccine programs for preventing disease caused by a California IBV field isolate.

text: Infectious bronchitis virus CA99 serotype was isolated from several broiler flocks in Northern California. The virus caused late-onset respiratory disease and increased airsacculitis condemnation in affected flocks despite the use of an established infectious bronchitis virus vaccination program. An experimental study compared Holland/Arkansas and Massachusetts/Arkansas vaccination protocols to determine the efficacy of commercial infectious bronchitis virus vaccines in reducing respiratory disease and airsacculitis lesions found at processing that were associated with a CA99 field isolate. All vaccination groups were given Massachusetts/Connecticut strains of infectious bronchitis virus vaccines at age 1 day followed by vaccination with either Holland/ Arkansas or Massachusetts/Arkansas vaccine strains at 18 days of age. Birds were challenged at age 31 days with a CA99 field isolate. Gross pathology, histopathology, and virus isolation were evaluated. Chickens vaccinated with Holland/Arkansas had marginally better protection against CA99 challenge than chickens vaccinated with Massachusetts/Arkansas, although differences were not statistically significant.


title: Interleukin-11: stimulation in vivo and in vitro by respiratory viruses and induction of airways hyperresponsiveness.

text: To address the role of IL-11 in viral airways dysfunction, we determined whether infectious agents that exacerbate asthma stimulate stromal cell IL-11 production, determined whether IL-11 could be detected at sites of viral infection and evaluated the effects of IL-11 on airway physiology. Respiratory syncytial virus (RSV), parainfluenza virus type 3 (PIV3), and rhinovirus (RV) 14 were potent stimulators while cytomegalovirus and adenovirus only weakly stimulated and herpes simplex virus type 2 and bacteria did not stimulate IL-11 elaboration. IL-11 was not detected or barely detected in nasal aspirates from children without, but was detected in aspirates from children with viral upper respiratory tract infections. The levels of IL-11 were highest in patients with clinically detectable wheezing. IL-11 also caused nonspecific airways hyperresponsiveness in BALB/c mice. These studies demonstrate that three major causes of viral-induced asthma, RSV, RV, and PIV, in contrast to other viruses and bacteria, share the ability to induce stromal cell IL-11 production. They also demonstrate that IL-11 can be detected in vivo during viral respiratory infections, that the presence of IL-11 correlates with clinical bronchospasm and that IL-11 is a potent inducer of airways hyperresponsiveness. IL-11 may be an important mediator in viral airways disorders.


ICES report: five policy recommendations from Toronto's SARS outbreak to improve the safety and efficacy of restrictions on hospital admissions to manage infectious disease outbreaks.


title: Droplet fate in indoor environments, or can we prevent the spread of infection?

text: UNLABELLED When considering how people are infected and what can be done to prevent the infections, answers from many disciplines are sought: microbiology, epidemiology, medicine, engineering, and physics. There are many pathways to infection spread, and among the most significant from the epidemiological point of view is airborne transport. Microorganisms can become airborne when droplets are generated during speech, coughing, sneezing, vomiting, or atomization of feces during sewage removal. The fate of the droplets is governed by the physical principles of transport, with droplet size being the most important factor affecting their dispersion, deposition on surfaces and determining the survival of microorganisms within the droplets. In addition, physical characteristics of the indoor environment as well as the design and operation of building ventilation systems are of critical importance. Do we understand the mechanisms of infection spread and can we quantify the droplet dynamics under various indoor conditions? Unfortunately no, as this aspect of infection spread has attracted surprisingly little scientific interest. However, investigations of numerous cases in which a large number of people were infected show how critical the physics of microorganism spread can be. This paper reviews the state of knowledge regarding mechanisms of droplet spread and solutions available to minimize the spread and prevent infections. PRACTICAL IMPLICATIONS Every day tens of millions of people worldwide suffer from viral infections of different severity at immense economic cost. There is, however, only minimal understanding of the dynamics of virus-laden aerosols, and so the ability to control and prevent virus spread is severely reduced, as was clearly demonstrated during the recent severe acute respiratory syndrome epidemic. This paper proposes the direction to significantly advance fundamental and applied knowledge of the pathways of viral infection spread in indoor atmospheric systems, through a comprehensive multidisciplinary approach and application of state-of-the-art scientific methods. Knowledge gained will have the potential to bring unprecedented economical gains worldwide by minimizing/reducing the spread of disease.


title: If you ask them, will they come? Predictors of quarantine compliance during a hypothetical avian influenza pandemic: results from a statewide survey.

text: BACKGROUND An influenza pandemic, such as that of the H1N1 virus, raises questions about how to respond effectively to a lethal outbreak. Most plans have focused on minimizing impact by containing the virus through quarantine, but quarantine has not been used widely in the United States and little is known about what would be the public's response. The purpose of this study was to investigate factors that influence an individual's decision to comply with a hypothetical avian influenza quarantine order. METHODS A total of 1204 adult Pennsylvania residents participated in a random digit dial telephone sample. The residents were interviewed regarding their attitudes about and knowledge of avian influenza and about compliance with quarantine orders, including staying at home or traveling to a government-designated facility. RESULTS Analysis of variance showed differences among demographic groups in willingness to comply with quarantine orders, with women and individuals not presently employed more willing to stay at home or to travel to a government-designated facility if ordered. Those who did not regularly attend religious services were significantly less willing than those who did attend regularly to comply with any type of quarantine order. Regression analysis indicated that demographic variables, overall knowledge of avian influenza, attitudes about its severity, and the belief that the respondent and/or his or her significant other(s) may contract it were predictive. CONCLUSIONS The results of this study can provide health planners and policy makers with information for improving their efforts to conduct a quarantine successfully, including crafting messages and targeting information to certain groups of people to communicate risk about the epidemic.


title: Retrospective analysis on Chinese patients diagnosed with acute renal failure hospitalized during the last decade (1994-2003).

text: OBJECTIVES To investigate the epidemiology, diagnosis and prognosis of acute renal failure (ARF) in hospitalized Chinese during the last decade. METHODS The diagnosis of patients with ARF in Peking University Third Hospital from January 1994 to December 2003 was reconfirmed and the data of epidemiology, etiology and prognosis were analyzed. RESULTS Only 209 discharged cases were diagnosed with ARF and all were reconfirmed. Two peak occurrences were found at ages of 35-45 and 60-80 with a male predominance of approximately 59.7%. Patients diagnosed with ARF accounted for 1.19 per thousand of the admissions in the same period and increased significantly in the last 5 years (p = 0.038). The creatinine level at diagnosis was 345.8 +/- 122.6 micromol/l and had no significant change (p > 0.05). The percent of hospital-acquired ARF (HA-ARF) demonstrated a significant increase in 1999-2003 compared to 1994-1998 (p = 0.008). Intrarenal ARF accounted for 73.69% and was multifactorial, with drugs, infections and operations as leading causes. Renal biopsy was performed in 37.32% (78/209) with 53.84% (42/78) having acute interstitial nephritis. Maintenance dialysis was discontinued in 46.41% because their renal function completely or partially recovered. The overall mortality was 37.91% without improvement over time. The mortality was 6.25% for patients in nephrology department, but 65.51% in ICU (p < 0.001), and was 21.6% for patients in community-acquired ARF (CA-ARF), but 63.1% in HA-ARF (p < 0.001). CONCLUSIONS During the past 10 years, the number of patients diagnosed with ARF has been rising in hospitalized Chinese. HA-ARF was the major source, and infections, drugs and operations were the leading causes. The diagnosis and prognosis of acute renal failure did not improve much in this population over the decade studied.


title: Pharmacies on the Frontline: Responding to the COVID-19 Pandemic.

text: The year 2020 will infamously be known for the COVID-19 pandemic. It certainly caught most of the population completely off-guard and forever changed their lives. Unfortunately, this pandemic is expected to make a resurgence in the fall, but hopefully more will be known about SARS-CoV-2, better treatment protocols will be established, and, possibly, a safe and effective vaccine will be developed and available soon. There are numerous drug shortages due to the COVID-19 pandemic. Some products are not commercially available in certain dosage forms needed by patients. Pharmacies (retail, independents, health systems) all over the U.S. are being asked to compound items such as hand sanitizers, saccharin solutions for FIT testing of N95 masks, and hydroxychloroquine suspension. The commercial lopinavir/ritonavir oral liquid is in short supply and may need to be compounded soon. We are all in this one together, from the essential workers to the children, and pharmacists are emerging as one of the first-line responders.


title: Clinical Characteristics and Blood Test Results in COVID-19 Patients.

text: OBJECTIVE An outbreak of pneumonia named COVID-19 caused by a novel coronavirus in Wuhan is rapidly spreading worldwide. The objective of the present study was to clarify further the clinical characteristics and blood parameters in COVID-19 patients. MATERIALS AND METHODS Twenty-three suspected patients and 64 patients with laboratory-confirmed SARS-Cov-2 infection were admitted to a designated hospital. Epidemiological, clinical, laboratory, and treatment data were collected and analyzed. RESULTS Of the 64 patients studied, 47 (73.4%) had been exposed to a confirmed source of COVID-19 transmission. On admission, the most common symptoms were fever (75%) and cough (76.6%). Twenty-eight (43.8%) COVID-19 patients showed leukopenia, 10 (15.6%) showed lymphopenia, 47 (73.4%) and 41 (64.1%) had elevated high-sensitivity C-reactive protein (hsCRP) and erythrocyte sedimentation rate (ESR), respectively, and 30 (46.9%) had increased fibrinogen concentration. After the treatment, the counts of white blood cells and platelets, and the level of prealbumin increased significantly, while aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and hsCRP decreased. COVID-19 patients with the hospital stay longer than 12 days had higher body mass index (BMI) and increased levels of AST, LDH, fibrinogen, hsCRP, and ESR. CONCLUSIONS Results of blood tests have potential clinical value in COVID-19 patients.


title: [A novel coronavirus, MERS-CoV].

text: A novel human coronavirus was identified in Saudi Arabia and Qatar as the causative agent of severe acute respiratory diseases in 2012. The virus was termed Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and is taken notice of important coronavirus caused severe diseases to human after the outbreak of severe acute respiratory syndrome (SARS) coronavirus. There is a lot of unknown characterization regarding MERS-CoV because of less than one year after finding the first case. MERS-CoV was related to the 2C betacoronavirus clade and is closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5. Thus, bats are thought to be natural hosts of this virus. Recently, there were reports supposed to be cases of human to human infection. There are growing concerns about spread of infection.


WHO investigates China's fall in SARS cases.


title: Readmission rates and life threatening events in COPD survivors treated with non-invasive ventilation for acute hypercapnic respiratory failure.

text: BACKGROUND Non-invasive ventilation (NIV) has been shown to reduce intubation and in-hospital mortality in patients with chronic obstructive pulmonary disease (COPD) and acute hypercapnic respiratory failure (AHRF). However, little information exists on the outcomes following discharge. A study was undertaken to examine the rates of readmission, recurrent AHRF, and death following discharge and the risk factors associated with them. METHODS A cohort of COPD patients with AHRF who survived after treatment with NIV in a respiratory high dependency unit was prospectively followed from July 2001 to October 2002. The times to readmission, first recurrent AHRF, and death were recorded and analysed against potential risk factors collected during the index admission. RESULTS One hundred and ten patients (87 men) of mean (SD) age 73.2 (7.6) years survived AHRF after NIV during the study period. One year after discharge 79.9% had been readmitted, 63.3% had another life threatening event, and 49.1% had died. Survivors spent a median of 12% of the subsequent year in hospital. The number of days in hospital in the previous year (p = 0.016) and a low Katz score (p = 0.018) predicted early readmission; home oxygen use (p = 0.002), APACHE II score (p = 0.006), and a lower body mass index (p = 0.041) predicted early recurrent AHRF or death; the MRC dyspnoea score (p<0.001) predicted early death. CONCLUSIONS COPD patients with AHRF who survive following treatment with NIV have a high risk of readmission and life threatening events. Further studies are urgently needed to devise strategies to reduce readmission and life threatening events in this group of patients.


title: Evaluation of a human group a rotavirus assay for on-site detection of bovine rotavirus.

text: Neonatal diarrhea induced by bovine group A rotavirus causes significant economic loss in the dairy and beef industry due to increased morbidity and mortality, treatment costs, and reduced growth rates. The objective of this study was to evaluate a human group A rotavirus assay (ImmunoCardSTAT Rotavirus [ICS-RV]) as an on-site diagnostic test for bovine rotavirus. When used with a collection of bovine diarrhea samples submitted to the Virology Section of the Diagnostic Center for Population and Animal Health at Michigan State University and compared to a bovine group A rotavirus-specific reverse transcription-PCR (RT-PCR), the ICS-RV assay had a sensitivity and specificity of 87.0 and 93.6%, respectively. A commercially available group A rotavirus enzyme-linked immunosorbent assay (ELISA) (Pathfinder; Sanofi Diagnostics, Redmond, Wash.), when used with the same fecal sample collection and compared to the same RT-PCR, had a sensitivity and specificity of 78.3 and 67.7%, respectively. Subsequently, the ICS-RV assay, RT-PCR, and a different commercially available group A rotavirus ELISA (Rotaclone; Meridian Diagnostics, Cincinnati, Ohio) were used to evaluate fecal samples collected from neonatal calves experimentally infected with bovine rotavirus. When diarrheic fecal samples that were positive for bovine rotavirus by RT-PCR were evaluated, the ICS-RV assay and the Rotaclone assay detected bovine rotavirus 85 and 95% of the time, respectively. Based on these studies, the ICS-RV assay appears to be an excellent test for detecting group A bovine rotaviruses. This assay may be useful as an on-site diagnostic test for veterinarians as an aid in the management of bovine neonatal diarrhea.


title: Respiratory Care Controversies II.

text: Despite a plethora of publications on the art and science of respiratory care, a number of basic issues remain unanswered. These clinical controversies are often settled by expert opinion and personal bias. By definition, a controversy has compelling arguments for each side of the issue. This Respiratory Care Journal Conference addressed 12 clinical controversies, ranging from spirometry screening for chronic lung disease to the timing of tracheostomy. This paper is a concise synopsis of the salient points of each side of each argument and provides the points of consensus and points of contention. When appropriate, further research is suggested to address still unanswered questions.


title: Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting.

text: Translational recoding of mRNA through a -1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of -1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of -1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.


title: Severe acute respiratory syndrome coronavirus gene 7 products contribute to virus-induced apoptosis.

text: The proteins encoded by gene 7 of the severe acute respiratory syndrome coronavirus (SARS-CoV) have been demonstrated to have proapoptotic activity when expressed from cDNA but appear to be dispensable for virus replication. Recombinant SARS-CoVs bearing deletions in gene 7 were used to assess the contribution of gene 7 to virus replication and apoptosis in several transformed cell lines, as well as to replication and pathogenesis in golden Syrian hamsters. Deletion of gene 7 had no effect on SARS-CoV replication in transformed cell lines, nor did it alter the induction of early apoptosis markers such as annexin V binding and activation of caspase 3. However, viruses with gene 7 disruptions were not as efficient as wild-type virus in inducing DNA fragmentation, as judged by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, indicating that the gene 7 products do contribute to virus-induced apoptosis. Disruption of gene 7 did not affect virus replication or morbidity in golden Syrian hamsters, suggesting that the gene 7 products are not required for acute infection in vivo. The data indicate that open reading frames 7a and 7b contribute to but are not solely responsible for the apoptosis seen in SARS-CoV-infected cells.


title: Thrombectomy without lysis: the future?

text: The results of the CaVent-study and the expected results of the ATTRACT and CAVA trials will form the base of evidence to support that rapid cloth removal is beneficial for patients with iliofemoral DVT. Although beneficial, there are still significant risks associated with this therapy. Therefore alternative methods for rapid cloth removal without lysis are a potential valid alternative. In this article we describe the techniques currently available for thrombolysis and discuss the potential improvements to be made to clot removal techniques in the future.


title: How severe acute respiratory syndrome (SARS) affected the department of anaesthesia at Singapore General Hospital.

text: The effects of the outbreak of severe acute respiratory syndrome (SARS) on the Singapore General Hospital's Department of Anaesthesia are described. Urgent measures to protect staff and patients were implemented. Clear direction in administration issues and clinical pathways with good logistic support were essential. Anaesthetists were at risk and anaesthetic practice had to change in view of strict infection control provisions. Contingency planning for future infectious diseases outbreaks is required. Lessons can be learnt by hospitals yet to be visited by this virulent infection.


title: Detection and isolation of coronavirus from feces of three herds of feedlot cattle during outbreaks of winter dysentery-like disease.

text: Clinical signs of a winter dysentery-like syndrome in 6- to 9-month-old cattle in 3 feedlots included acute onset of diarrhea with high morbidity and low mortality, respiratory tract problems that included dyspnea, coughing, and nasal discharge, and high rectal temperatures. Bovine coronavirus was detected by use of an ELISA and immune electron microscopy in fecal and nasal swab samples and by immunohistochemical analysis of intestinal sections collected from calves during necropsy. Bovine coronavirus should be considered in the differential diagnoses for diseases that cause acute onset of bloody diarrhea in feedlot cattle.


title: Isolation and characterization of a coronavirus from elk calves with diarrhea.

text: This is the first report of the isolation of a coronavirus from elk calves. Two fecal samples from elk calves with diarrhea were shown to be positive for coronavirus-like particles by electron microscopy, and the particles were propagated in the human rectal tumor-18 cell line. After 24 h, syncytia were observed, and cell culture supernatants from both samples showed hemagglutinating activity with mouse erythrocytes. Cells infected with both elk coronavirus (ECV) isolates reacted with Z3A5, a monoclonal antibody against the spike protein of bovine coronavirus (BCV), on an indirect fluorescent antibody test. The protein profiles of both ECV isolates were similar to that of BCV as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. On Northern blot analysis, the transcriptional pattern of ECV was typical of coronaviruses, with a nested set of transcripts with common 3' end sequences. Based on a published nucleoprotein gene sequence for BCV (Mebus isolate), we arbitrarily designed two primers for amplification by PCR. After cloning, the nucleoprotein was sequenced and a high degree of homology (99%) between the nucleoprotein gene sequences of ECV and BCV was observed. Thus, ECV is closely related genetically and antigenically to BCV and will be a new member of antigenic group 2 of the mammalian coronaviruses, which possess hemagglutinin-esterase protein.


title: Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlândia, MG, Brazil.

text: The main viruses involved in acute respiratory diseases among children are: respiratory syncytial virus (RSV), influenzavirus (FLU), parainfluenzavirus (PIV), adenovirus (AdV), human rhinovirus (HRV), and the human metapneumovirus (hMPV). The purpose of the present study was to identify respiratory viruses that affected children younger than five years old in Uberlândia, Midwestern Brazil. Nasopharyngeal aspirates from 379 children attended at Hospital de Clínicas (HC/UFU), from 2001 to 2004, with acute respiratory disease, were collected and tested by immunofluorescence assay (IFA) to detect RSV, FLU A and B, PIV 1, 2, and 3 and AdV, and RT-PCR to detect HRV. RSV was detected in 26.4% (100/379) of samples, FLU A and B in 9.5% (36/379), PIV 1, 2 and 3 in 6.3% (24/379) and AdV in 3.7% (14/379). HRV were detected in 29.6% (112/379) of the negative and indeterminate samples tested by IFI. RSV, particularly among children less than six months of life, and HRV cases showed highest incidence. Negative samples by both IFA and RT-PCR might reflect the presence of other pathogens, such as hMPV, coronavirus, and bacteria. Laboratorial diagnosis constituted an essential instrument to determine the incidence of the most common viruses in respiratory infections among children in this region.


title: Quantitation of intestinal D-xylose absorption in normal broilers and in broilers with pale-bird syndrome.

text: A micromethod was used in order to quantitate intestinal D-xylose absorption in young and extremely small birds. This test was performed in broilers collected from two farms from which birds were extremely uneven in body size and were passing poorly digested or undigested feed. A similar syndrome had been seen on all grow-outs during the 6 months before this investigation. Broilers were also collected and tested from three farms where no clinical signs of disease were seen. D-Xylose absorption peaks and curves for normal broilers closely resembled those observed in normal humans. Mean plasma D-xylose concentrations for virus-infected broilers and for broilers with pale-bird syndrome were consistently lower than concentrations for normal broilers (P = 0.009). Reoviruses, small coronavirus-like particles, small round virus particles, and abundant bacterial flagellar fragments were seen in fecal samples from broilers with pale-bird syndrome. Production performance was lowest on farms showing clinical signs of this syndrome.


title: Pathogenesis of chimeric MHV4/MHV-A59 recombinant viruses: the murine coronavirus spike protein is a major determinant of neurovirulence.

text: The mouse hepatitis virus (MHV) spike glycoprotein, S, has been implicated as a major determinant of viral pathogenesis. In the absence of a full-length molecular clone, however, it has been difficult to address the role of individual viral genes in pathogenesis. By using targeted RNA recombination to introduce the S gene of MHV4, a highly neurovirulent strain, into the genome of MHV-A59, a mildly neurovirulent strain, we have been able to directly address the role of the S gene in neurovirulence. In cell culture, the recombinants containing the MHV4 S gene, S4R22 and S4R21, exhibited a small-plaque phenotype and replicated to low levels, similar to wild-type MHV4. Intracranial inoculation of C57BL/6 mice with S4R22 and S4R21 revealed a marked alteration in pathogenesis. Relative to wild-type control recombinant viruses (wtR13 and wtR9), containing the MHV-A59 S gene, the MHV4 S gene recombinants exhibited a dramatic increase in virulence and an increase in both viral antigen staining and inflammation in the central nervous system. There was not, however, an increase in the level of viral replication in the brain. These studies demonstrate that the MHV4 S gene alone is sufficient to confer a highly neurovirulent phenotype to a recombinant virus deriving the remainder of its genome from a mildly neurovirulent virus, MHV-A59. This definitively confirms previous findings, suggesting that the spike is a major determinant of pathogenesis.


Substrate specificity and rational design of peptidomimetic inhibitors for SARS coronavirus main protease.


title: Murine coronavirus gene 1 polyprotein contains an autoproteolytic activity.

text: The 5' most gene of the murine coronavirus genome, gene 1, is presumed to encode the viral RNA-dependent RNA polymerase. cDNA clones representing this gene encompass more than 22 kilobases, suggesting that this region may encode multifunctional polyprotein(s). It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To identify possible functional domains of gene 1 and further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of the murine coronavirus mouse hepatitis virus strain JHM were subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. Although p28 is encoded from the first 1 kilobase at the 5'-end of the genome, translation of in vitro transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3 kilobase region was synthesized. This result suggests that the region close to 5.3 kilobases from the 5'-end of the genomic RNA is essential for the proteolytic cleavage and may contain an autoproteolytic activity. Addition of the protease inhibitor ZnCl2 blocked cleavage of the p28 protein. Site-directed mutagenesis of Cys residue 1137 significantly reduced the cleavage of the p28 protein, indicating that this residue, probably in conjunction with a downstream domain, plays an essential role in the cleavage of p28. This Cys residue may be part of a papain-like autoprotease encoded by gene 1.


title: The challenge of providing holistic care in a viral epidemic: opportunities for palliative care.

text: OBJECTIVE To determine, using the perspective of a hospital-based palliative care service (PCS), the spiritual and psychosocial impact of a novel and potentially fatal viral epidemic on patients, their families and health care workers. DESIGN Qualitative study using semi-structured interviews. The data were analysed using the constant comparative method and were validated using respondent validation. PARTICIPANTS Eight palliative care workers. SETTING Tan Tock Seng Hospital (TTSH), the hospital designated to manage all cases of Severe Acute Respiratory Syndrome (SARS) in Singapore. RESULTS Disease containment resulted in isolation, where isolation meant the disruption of connectedness. This disruption of connectedness took place at multiple levels: in the patient himself, between the patient and the family, within the family, between the patient and the health care worker, between the patient and society, and between health care workers. As the nature of the disease was uncertain, prognostication was inaccurate. This created difficulties in helping patients and their families prepare for death. The fear of facing the unknown led to safety-seeking behaviours, which could be overcome by repeated exposure to the feared situation. The process of bereavement was disturbed, as traditional death rituals could not be performed by the family. Informants perceived themselves to be suffering as they suffered the same anxieties, fears and grief as the patient. CONCLUSIONS Dealing with a novel viral epidemic creates spiritual and psychosocial issues similar to those encountered in a palliative care practice. Palliative care workers would do well to be aware of such issues and act proactively when such epidemics arise.


title: Next generation sequencing technologies: tool to study avian virus diversity.

text: Increased globalisation, climatic changes and wildlife-livestock interface led to emergence of novel viral pathogens or zoonoses that have become serious concern to avian, animal and human health. High biodiversity and bird migration facilitate spread of the pathogen and provide reservoirs for emerging infectious diseases. Current classical diagnostic methods designed to be virus-specific or aim to be limited to group of viral agents, hinder identifying of novel viruses or viral variants. Recently developed approaches of next-generation sequencing (NGS) provide culture-independent methods that are useful for understanding viral diversity and discovery of novel virus, thereby enabling a better diagnosis and disease control. This review discusses the different possible steps of a NGS study utilizing sequence-independent amplification, high-throughput sequencing and bioinformatics approaches to identify novel avian viruses and their diversity. NGS lead to the identification of a wide range of new viruses such as picobirnavirus, picornavirus, orthoreovirus and avian gamma coronavirus associated with fulminating disease in guinea fowl and is also used in describing viral diversity among avian species. The review also briefly discusses areas of viral-host interaction and disease associated causalities with newly identified avian viruses.


title: The use of PCR genome mapping for the characterisation of TGEV strains.

text: Previous studies on different transmissible gastroenteritis virus (TGEV) strains, including porcine respiratory coronavirus (PRCV), have identified regions within the genome that are polymorphic as regards insertions and deletions. For example the 672 base deletion within the S gene and multiple deletions 5', within and 3' of the ORF-3a gene were detected in strains of PRCV. The presence of deletions may be associated with a change in the virulence, attenuation or tissue tropism of the isolate. The Nouzilly (188-SG) TGEV vaccine strain was attenuated by passage of a cell culture adapted virulent isolate D-52 188 times through swine testis cells after treatment with gastric juice. PCR amplification with oligonucleotides, corresponding to known TGEV sequences, were used to analyse D-52 and 188-SG for genetic variation. Results with several pairs of oligonucleotides within the first 1565 nucleotides of the S gene did not identify a deletion within this region of the genome from either strain. However, oligonucleotides directed against the ORF-3a/3b region detected a deletion of about 250 nucleotides within the 188-SG genome but not in the D-52 genome. Since all the attenuated TGEV strains so far sequenced, PRCV, Miller SP and 188-SG, contained deletions within the ORF-3a/3b, it would suggest that this region of the TGEV genome is involved in regulating viral virulence.


Functional role of ICAM-3 polymorphism in genetic susceptibility to SARS infection.


title: RNA interference--significance and applications.

text: RNA interference (RNAi) is a post-transcriptional, highly conserved process in eukaryotes that leads to specific gene silencing through degradation of the target mRNA. This mechanism is mediated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene. The dsRNA is processed into small interfering RNA (siRNA) by an enzyme called Dicer, and the siRNAs are then incorporated into a multi-component RNA-induced silencing complex, which finds and cleaves the target mRNA. In plants and worms, amplification of the silencing signal and cell-to-cell RNAi spreading is observed. The proposed biological roles of RNAi include resistance to viruses, transposons (mainly in plants), and the silencing and regulation of gene expression, particularly during development. In developmental gene control, specific small RNAs (micro RNA and small temporal RNA) are involved, which are processed in the same way as dsRNAs but act at the level of translation. RNAi technology has become a powerful tool in functional genomic analyses and may prove to be a useful method to develop highly specific gene-silencing therapeutics against viral infections and cancer in the future.


title: Evaluation of quantitative latex agglutination for detection of Cryptosporidium parvum, E. coli K99, and rotavirus in calf feces.

text: A new methodology for detection of rotavirus, Escherichia coli K99, and Cryptosporidium parvum in bovine fecal samples was developed based on a quantitative latex agglutination technique (QLAT). Calibrated microspheres coated with specific antibodies to 1 of the enteric pathogens are quantitatively agglutinated by the antigens present in diluted fecal sample. The test is performed in a 96-well flat-bottom plate. The samples were tested with a commercial enzyme-linked immunosorbent assay kit prior to being analyzed by QLAT. The calculated sensitivity and specificity are adequate for field conditions, because the amount of the pathogenic agents is generally high. The overall time to perform the test was about 20 minutes.


title: [Recombination between RNA genomes].

text: Evidence for the intermolecular recombination between the RNA genomes of picornaviruses and coronaviruses as well as current models of the mechanisms of these phenomena are reviewed. Biological implications of the recombination between RNA genomes are briefly discussed. Examples of the recombinant analysis of the viral genome functions are given.