Detection of Entebbe Bat Virus after 54 Years

Entebbe bat virus (ENTV; Flaviviridae: Flavivirus), closely related to yellow fever virus, was first isolated from a little free-tailed bat (Chaerephon pumilus) in Uganda in 1957, but was not detected after that initial isolation. In 2011, we isolated ENTV from a little free-tailed bat captured from the attic of a house near where it had originally been found. Infectious virus was recovered from the spleen and lung, and the viral RNA was sequenced and compared with that of the original isolate. Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Further study of this virus would provide valuable insights into the ecological and genetic factors governing the evolution and transmission of bat- and mosquito-borne flaviviruses.Bat virus investigations in Uganda were initially inspired in the mid-1950s by the isolations of rabies virus and a novel flavivirus, later to become known as Rio Bravo virus (Flaviviridae: Flavivirus), from the salivary glands of insectivorous bats in the United States.^1–3 Subsequently, investigators at the Uganda Virus Research Institute (UVRI) collected bats from the attic of UVRI, dissected salivary glands, and isolated viruses by intracerebral inoculation of triturated salivary gland extracts into neonatal mice.^4,5 This first effort resulted in the isolation of Entebbe bat salivary gland virus, strain IL-30 (Flaviviridae: Flavivirus) from a little free-tailed bat (Chaerephon pumilus) (Cretzchmar).^4,5 The taxon was then recognized by one of its synonyms: Tadarida (Chaerephon) limbata (Peters). Lumsden and others^4,5 determined that this isolate was a novel flavivirus through a series of neutralization tests using immune sera against 20 arboviruses as well as against Rio Bravo virus. By 1964, bat virus research had expanded at UVRI, and sampling of 1,022 additional bats yielded 14 strains of viruses including Dakar bat virus (Flaviviridae: Flavivirus) also from little free-tailed bats, and Bukalasa bat virus (Flaviviridae: Flavivirus) from Angolan free-tailed bats (Mops condylurus) (A. Smith).⁶ Isolation of Mount Elgon bat virus (Rhabdoviridae: Ledantevirus) followed shortly thereafter from an eloquent horseshoe bat (Rhinolophus hildebrandtii eloquens) (K. Anderson) captured in Kenya, with the virus being isolated and characterized at UVRI.^7,8 Additional bat virus discoveries in Uganda later included Kasokero virus (Bunyaviridae, unassigned) from an Egyptian fruit bat (Rousettus aegyptiacus) (E. Geoffroy).⁹ Entebbe bat virus (ENTV, renamed), Bukalasa bat virus, and Dakar bat virus were all isolated from insectivorous bats captured in the attic of UVRI.¹⁰ Serological surveys suggested that the infection prevalence of ENTV in wild populations of little free-tailed bats was high, but ENTV was never isolated again after the original discovery.^10,11To follow up on these early studies, arbovirus surveillance of bats was conducted in Uganda from 2011 to 2013. Bats were captured from attics in several locations around Entebbe and Kampala as part of a larger, country-wide sampling effort to be reported elsewhere. All bat captures were conducted under the approval of CDC/DVBD IACUC protocol 010-015. Bats were captured in attics using mist nets, taking appropriate biosafety precautions. On capture, bats were placed individually in cloth holding bags. Bats were anesthetized with halothane, exsanguinated by cardiac puncture, and euthanized by halothane overdose and cervical dislocation. Blood from bats was collected directly into serum separator tubes, centrifuged in the field, and placed immediately in a nitrogen dry vapor shipper. Liver, spleen, heart, lung, and kidney were all collected directly into cryovials and stored immediately on dry ice. In total, 95 little free-tailed bats and 34 Angolan free-tailed bats were captured in 2011, 15 little free-tailed bats in 2012, and 70 little free-tailed bats in 2013.Virus isolation was attempted first from spleens, and on a positive result, virus isolation was also performed from the remaining tissues harvested from the infected bat. A single virus was isolated from 1 of 180 (0.5%) little free-tailed bats. The infected bat was an adult male, captured in Banga, Nakiwogo (0°04.884′ N, 32°27.030′ E) on June 23, 2011, near UVRI. Infectious virus was isolated from the spleen and lung; no infectious virus was recovered from the heart, liver, or kidney. No other tissues, oral or fecal swabs were collected from the insectivorous bats captured in 2011. The isolate was initially identified as ENTV by sequencing of the flavivirus NS5 amplicon using primers FU2/CFD3,¹² followed by next-generation sequencing (NGS). Virus isolations, nucleic acid preparations, and NGS were performed as described previously.¹³ Novel primers were designed from contigs generated from the NGS data using Primer 3 software (Whitehead Institute for Biomedical Research, Cambridge, MA)¹⁴ (Supplemental Table 1), and the sequence for the entire genome was confirmed by direct sequencing. End terminal sequences were obtained using 5′/3′ RACE (FirstChoice^® RLM-RACE, Life Technologies, Carlsbad, CA) according to the manufacturer''s instructions. Virus-specific RACE primers are provided in Supplemental Table 1. The 5′ outer reaction used primer 514R in combination with the 5′ RACE outer primer. The 5′ RACE inner reactions used primer 388R together with the 5′ RACE inner primer; amplification was also achieved with primers 15F + 388R. The 3′ outer reaction used either primer 9882F or 10328F in combination with the 3′ RACE outer primer. The 3′ inner RACE was not successful. A maximum likelihood phylogenetic tree was generated in MEGA version 6.0¹⁵ (Center for Evolutionary Medicine and Informatics, Biodesign Institute, Tempe, AZ) using 10,493 nucleotides spanning the open reading frame sequence of select flavivirus genomes available in GenBank (Figure 1 ). The analysis was performed using a complete deletion substitution model and 1,000 bootstrap replicates.Open in a separate windowFigure 1.Maximum likelihood tree based on open reading frame sequence (complete gap deletion). Scale bar indicates nucleotide substitutions per site. Bootstrap values > 50% are shown (1,000 replicates). CFAV = cell fusing agent virus (NC001564); DENV1 = dengue type 1 ({"type":"entrez-nucleotide","attrs":{"text":"M87512","term_id":"323660","term_text":"M87512"}}M87512); DENV2 = dengue type 2 ({"type":"entrez-nucleotide","attrs":{"text":"M20558","term_id":"323449","term_text":"M20558"}}M20558); DENV3 = dengue type 3 ({"type":"entrez-nucleotide","attrs":{"text":"M93130","term_id":"323468","term_text":"M93130"}}M93130); DENV4 = dengue type 4 ({"type":"entrez-nucleotide","attrs":{"text":"AF326573","term_id":"12659201","term_text":"AF326573"}}AF326573); EHV = Edge Hill virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859060","term_id":"146411782","term_text":"DQ859060"}}DQ859060); ENTV = Entebbe bat virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641); RBV = Rio Bravo virus ({"type":"entrez-nucleotide","attrs":{"text":"JQ582840","term_id":"383215103","term_text":"JQ582840"}}JQ582840); SEPV = Sepik virus (NC008719); SLEV = St. Louis encephalitis virus (NC007580); SOKV = Sokoluk virus ({"type":"entrez-nucleotide","attrs":{"text":"NC_026624","term_id":"765702601","term_text":"NC_026624"}}NC_026624); TBEV = tick-borne encephalitis virus (NC001672); WNV = West Nile virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ211652","term_id":"77166600","term_text":"DQ211652"}}DQ211652); WSLV = Wesselsbron virus ({"type":"entrez-nucleotide","attrs":{"text":"DQ859058","term_id":"146411778","term_text":"DQ859058"}}DQ859058); YFV = yellow fever virus ({"type":"entrez-nucleotide","attrs":{"text":"K02749","term_id":"336192","term_text":"K02749"}}K02749); YOKV = Yokose virus (NC005039); ZIKV = Zika virus ({"type":"entrez-nucleotide","attrs":{"text":"AF013415","term_id":"2731554","term_text":"AF013415"}}AF013415).In total, 10,663 nucleotides of the ENTV genome were obtained (GenBank Accession number {"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893), of which the polypeptide is encoded between nucleotides 120 and 10,355. Kuno and Chang¹⁶ published the only other existing ENTV genome ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641) sequenced from the original IL-30 strain. Compared with that original sequence, we report an additional 153 nucleotides in the 3′ noncoding region, but also experienced difficulty in obtaining the complete 3′ terminal sequence.¹⁶ Across the polypeptide sequence, there were 76 amino acid substitutions, resulting in 97.8% identity at the amino acid level between the 1957 and 2011 isolates. Before sequencing, the 1957 isolate had been passaged in suckling mouse brain, but the earlier passage history is unclear; the 2011 isolate was passaged twice in Vero cells. This 2.2% amino acid divergence is within the range that has been observed for other flaviviruses with isolations ranging between 1 and 49 years apart,^17–19 indicating relatively little change in the ENTV genome in 54 years since the first isolation.Every gene had at least one amino acid change except for PrM and M (20 It has also been shown to associate with host cellular vimentin during dengue virus replication to stabilize the replication complex.²¹ The functional significance of this high percentage of amino acid divergence has yet to be determined, but Hahn and others²² surmised that a large number of amino acid substitutions could be accommodated among the small, hydrophobic polypeptides NS2A, NS2B, NS4A, and NS4B so long as the hydrophobicity profile remained unchanged. In this case, changes to the NS4A hydrophobicity profile between these two isolates were inconsequential. Two of the 76 amino acid changes occurred at the junctions of NS2A/NS2B, and at NS4A/2K. The lengths of all of the genes were identical to those previously reported.¹⁶

Table 1

Length of each gene region in the Entebbe bat virus genome and the number (%) of amino acid (aa) substitutions in the 2011 isolate ({"type":"entrez-nucleotide","attrs":{"text":"KP233893","term_id":"873167403","term_text":"KP233893"}}KP233893) relative to the 1957 isolate ({"type":"entrez-nucleotide","attrs":{"text":"DQ837641","term_id":"112818954","term_text":"DQ837641"}}DQ837641)

Urine Collagen Fragments and CKD Progression—The Cardiovascular Health Study

Tubulointerstitial fibrosis is common with ageing and strongly prognostic for ESRD but is poorly captured by eGFR or urine albumin to creatinine ratio (ACR). Higher urine levels of procollagen type III N-terminal propeptide (PIIINP) mark the severity of tubulointerstitial fibrosis in biopsy studies, but the association of urine PIIINP with CKD progression is unknown. Among community-living persons aged ≥65 years, we measured PIIINP in spot urine specimens from the 1996 to 1997 Cardiovascular Health Study visit among individuals with CKD progression (30% decline in eGFR over 9 years, n=192) or incident ESRD (n=54) during follow-up, and in 958 randomly selected participants. We evaluated associations of urine PIIINP with CKD progression and incident ESRD. Associations of urine PIIINP with cardiovascular disease, heart failure, and death were evaluated as secondary end points. At baseline, mean age (±SD) was 78±5 years, mean eGFR was 63±18 ml/min per 1.73 m², and median urine PIIINP was 2.6 (interquartile range, 1.4–4.2) μg/L. In a case-control study (192 participants, 231 controls), each doubling of urine PIIINP associated with 22% higher odds of CKD progression (adjusted odds ratio, 1.22; 95% confidence interval, 1.00 to 1.49). Higher urine PIIINP level was also associated with incident ESRD, but results were not significant in fully adjusted models. In a prospective study among the 958 randomly selected participants, higher urine PIIINP was significantly associated with death, but not with incident cardiovascular disease or heart failure. These data suggest higher urine PIIINP levels associate with CKD progression independently of eGFR and ACR in older individuals.     

Anesthesia and the developing brain: a way forward for clinical research

It is now well established that many general anesthetics have a variety of effects on the developing brain in animal models. In contrast, human cohort studies show mixed evidence for any association between neurobehavioural outcome and anesthesia exposure in early childhood. In spite of large volumes of research, it remains very unclear if the animal studies have any clinical relevance; or indeed how, or if, clinical practice needs to be altered. Answering these questions is of great importance given the huge numbers of young children exposed to general anesthetics. A recent meeting in Genoa brought together researchers and clinicians to map a path forward for future clinical studies. This paper describes these discussions and conclusions. It was agreed that there is a need for large, detailed, prospective, observational studies, and for carefully designed trials. It may be impossible to design or conduct a single study to completely exclude the possibility that anesthetics can, under certain circumstances, produce long‐term neurobehavioural changes in humans; however , observational studies will improve our understanding of which children are at greatest risk, and may also suggest potential underlying etiologies, and clinical trials will provide the strongest evidence to test the effectiveness of different strategies or anesthetic regimens with respect to better neurobehavioral outcome.     

The effect of tranexamic acid on artificial joint materials: a biomechanical study (the bioTRANX study)

Background

Tranexamic acid (TXA) has been successfully used to reduce bleeding in joint replacement. Recently local TXA has been advocated to reduce blood loss in total knee or hip replacement; however, this raised concerns about potential adverse effects of TXA upon the artificial joint replacement.

Materials and methods

In this biomechanical study we compared the effects of TXA and saline upon the following biomechanical properties of artificial joint materials—(1) tensile properties (ultimate strength, stiffness and Young’s modulus), (2) the wear rate using a multi-directional pin-on-plate machine, and (3) the surface topography of pins and plates before and after wear rate testing.

Results

There were no significant differences in tensile strength, wear rates or surface topography of either ultra-high-molecular-weight polyethylene pins or cobalt chromium molybdenum metal plates between specimens soaked in TXA and specimens soaked in saline.

Conclusion

Biomechanical testing shows that there are no biomechanical adverse affects on the properties of common artificial joint materials from using topical TXA.

Level of evidence

V     

The Kaiser Permanente Shoulder Arthroplasty Registry: Results from 6,336 primary shoulder arthroplasties

Background and purpose

Shoulder arthroplasty is being performed in the United States with increasing frequency. We describe the medium-term findings from a large integrated healthcare system shoulder arthroplasty registry.

Patients and methods

Shoulder arthroplasty cases registered between January 2005 and June 2013 were included for analysis. The registry included patient characteristics, surgical information, implant data, attrition, and patient outcomes such as surgical site infections, venous thromboembolism, and revision procedures.

Results

During the study period, 6,336 primary cases were registered. Median follow-up time for all primaries was 3.3 years; 461 cases were lost to follow-up by ending of health plan membership. Primary cases were predominantly female (56%) and white (81%), with an average age of 70 years. The most common reason for surgery was osteoarthritis in 60% of cases, followed by acute fracture (17%) and rotator cuff tear arthropathy (15%). In elective shoulder arthroplasty procedures, 200 all-cause revisions (4%) were reported, with glenoid wear being the most common reason.

Interpretation

Most arthroplasties were elective procedures: over half performed for osteoarthritis. Glenoid wear was the most common reason for revision of primary shoulder arthroplasty in elective cases.In the period 2000 through 2010, over 200,000 shoulder arthroplasties were performed in the USA for osteoarthritis (Trofa et al. 2014). With the increasing use of shoulder arthroplasty (SA) over the past decade (Kim et al. 2011) and projections that future growth rates of SA may exceed those of hip and knee arthroplasty (Day et al. 2010), the need to track the outcomes of SA is becoming increasingly important.Arthroplasty registries provide an important mechanism for tracking surgical outcomes. In the fields of total hip arthroplasty and total knee arthroplasty, registries have demonstrated their importance in monitoring revisions, complications, and mortality, identifying outlier prostheses, and improving quality of care (Graves et al. 2004, Herberts and Malchau 2000, de Steiger et al. 2013, Paxton et al. 2010). SA registries have also provided critical information about demographics, survival, and outlier implants, though there have been considerably fewer publications from the younger national SA registries than from the more established hip and knee registries (Clitherow et al. 2014, Young et al. 2013, Rasmussen et al. 2012a, Rasmussen et al. 2014a and b, Fevang et al. 2009, Fevang et al. 2013). The lack of a national US registry emphasizes the need to use existing US registries to conduct international comparisons of SA patients, implants, surgical techniques, and outcomes. We present the medium-term findings of a large integrated healthcare system SA registry.     

Idiopathic Flushing with Dysesthesia: Treatment with the 585nm Pulsed Dye Laser

Objective: The purpose of this study was to analyze the efficacy and safety of the 585nm pulsed dye laser for the treatment of idiopathic flushing with dysesthesia. Design: This was a retrospective study of patients treated with a 585nm pulsed dye laser with fluences ranging from 3.5 to 7.5J/cm² (purpura threshold fluences), a pulse duration of 450μsec, and a spot size of 5 or 10mm. Setting: The Ronald 0. Perelman Department of Dermatology at New York University Medical Center. Participants: Ten adult subjects who presented with flushing with dysesthesia. Measurements: Participants subjectively evaluated the decrease in dysesthesia and the number of flushing episodes. The objective response to treatment was evaluated by a single physician using pre- and postoperative photographs. The severity of postoperative erythema was compared with baseline using an ordinal scale ranging from zero (resolution of erythema) to four (76-100% of baseline erythema). Results: The mean number of treatments received by the subjects was seven. The mean fluence was 6.66J/cm². Subjectively, 100 percent of subjects reported a decrease in dysethesia and the number of flushing episodes. Objectively, subjects demonstrated at least a 62.5-percent reduction in erythema. Conclusion: Laser surgery provided subjective relief of dysesthesia and decreased the number of flushing episodes with a greater than 62-percent objective reduction in the severity of erythema. The 585nm pulsed dye laser is a safe, efficacious treatment for the signs and symptoms of idiopathic flushing with dysesthesia.Flushing is a transient, episodic redness of the face, neck, upper chest, and/or epigastric area that is associated with certain diseases, ingestion of certain drugs or other substances, heat, emotional factors, or physical exertion.1 Blushing is flushing exclusively provoked by an emotional stimulus.2,3 Dysesthesia is defined as an unpleasant, abnormal sensation that is produced by normal stimuli.4Idiopathic flushing is a diagnosis of exclusion.3,5-8 In a subset of patients with idiopathic flushing, dysesthesia may be noted, and patients describe this symptom as a warm, unpleasant, burning sensation. Patients with this constellation of signs and symptoms, which is henceforth referred to as idiopathic flushing with dysesthesia, consistently deny concomitant pruritus. Furthermore, associated signs and symptoms, such as bronchospasm, abdominal cramps, diarrhea, headache, hypotension, or tachycardia, are rare in these patients.The objectives of this study were to define the characteristics of a poorly defined disorder, idiopathic flushing with dysesthesia, and to evaluate the treatment of subjects with this disorder using the 585nm pulsed dye laser.     

Efficacy and safety of rofecoxib 12.5 mg versus nabumetone 1,000 mg in patients with osteoarthritis of the knee: a randomized controlled trial

OBJECTIVES: To evaluate the use of starting doses of rofecoxib and nabumetone in patients with osteoarthritis (OA) of the knee. DESIGN: A 6-week, randomized, parallel-group, double-blind, placebo-controlled study. SETTING: One hundred thirteen outpatient sites in the United States. PARTICIPANTS: A total of 1,042 male and female patients aged 40 and older with OA of the knee (>6 months). INTERVENTIONS: Rofecoxib 12.5 mg once a day (n=424), nabumetone 1,000 mg once a day (n=410), or placebo (n=208) for 6 weeks. MEASUREMENTS: The primary efficacy endpoint was patient global assessment of response to therapy (PGART) over 6 weeks, which was also specifically evaluated over the first 6 days. The main safety measure was adverse events during the 6 weeks of treatment. RESULTS: The percentage of patients with a good or excellent response to therapy as assessed using PGART at Week 6 was significantly higher with rofecoxib (55.4%) than nabumetone (47.5%; P=.018) or placebo (26.7%; P<.001 vs rofecoxib or nabumetone). Median time to first report of a good or excellent PGART response was significantly shorter in patients treated with rofecoxib (2 days) than with nabumetone (4 days, P=.002) and placebo (>5 days, P<.001) (nabumetone vs placebo; P=.007). The safety profiles of rofecoxib and nabumetone were generally similar, including gastrointestinal, hypertensive, and renal adverse events. CONCLUSION: Rofecoxib 12.5 mg daily demonstrated better efficacy over 6 weeks of treatment and quicker onset of OA efficacy over the first 6 days than nabumetone 1,000 mg daily. Both therapies were generally well tolerated.     

Ruptured aneurysm of the right sinus of valsalva associated with a ventricular septal defect and an anomalous coronary artery

Aneurysms of the sinus of Valsalva are extremely rare. Ruptured aneurysms of the sinus of Valsalva are frequently associated with other congenital defects, particularly with ventricular septal defect, aortic valve regurgitation, and bicuspid aortic valve. We describe the case of a 26-year-old man who had a ruptured aneurysm of the right coronary sinus, a ventricular septal defect, and an anomalous origin of the right coronary artery. Successful surgical correction of the aneurysm and ventricular septal defect was performed with patch repair and aortic valve replacement. A review of the English-language medical literature revealed only 1 other case of a sinus of Valsalva aneurysm associated with a ventricular septal defect and an anomalous coronary artery. Previously published reports of the coexistence of a single coronary artery with a sinus of Valsalva aneurysm or with a ventricular septal defect, and their management, are discussed herein.     

Review of Methods for Studying Maturation of Human Erythroblasts in Vitro: Evaluation of a New Method of Culture of Cell Suspensions in a Clot-Free Medium

Characteristics of various tissue and cell culture methods for studyingmaturation of human erythroblasts were reviewed and the basic assumptions required in their use were examined.

The relationship of maturation and proliferation of erythroblasts in different types of cultures was considered.

Criteria for selection of an in vitro model for studying maturation wereestablished. A new clot-free culture system in roller tubes was found tobe superior to those previously described because of convenience, replicabilityand adaptability to studies of proliferation.

The results of challenging the new culture system with various typesof cell-plasma relationships indicated that it was effective in distinguishingamong erythroblasts and plasmas of iron deficiency anemia, thalassemia,azotemia and erythrocytosis, and that the deductions arrived at were similarto those derived from the use of less convenient in vitro systems in whichdifferent parameters of maturation were used.

Experience with the new method suggests that it may be useful in thestudy of humoral factors affecting maturation of human erythroblasts.

The chief limitation of the proposed method, as for other in vitro methods,is that only direct effects of humoral agents on erythroblasts can be studied.

Submitted on December 17, 1958 Accepted on February 21, 1959