title: Estimation of risk factors for COVID-19 mortality - preliminary results

text: Since late December 2019 a new epidemic outbreak has emerged from Whuhan, China. Rapidly the new coronavirus has spread worldwide. China CDC has reported results of a descriptive exploratory analysis of all cases diagnosed until the 11th February 2020, presenting the epidemiologic curves and geo-temporal spread of COVID-19 along with case fatality rate according to some baseline characteristics, such as age, gender and several well-established high prevalence comorbidities. Despite this, we intend to increase even further the predictive value of that manuscript by presenting the odds ratio for mortality due to COVID-19 adjusted for the presence of those comorbidities and baseline characteristics such as age and gender. Besides, we present a way to determine the risk of each particular patient, given his characteristics. We found that age is the variable that presents higher risk of COVID-19 mortality, where 60 or older patients have an OR = 18.8161 (CI95%[7.1997; 41.5517]). Regarding comorbidities, cardiovascular disease appears to be the riskiest (OR=12.8328 CI95%[10.2736; 15.8643], along with chronic respiratory disease (OR=7.7925 CI95%[5.5446; 10.4319]). Males are more likely to die from COVID-19 (OR=1.8518 (CI95%[1.5996; 2.1270]). Some limitations such as the lack of information about the correct prevalence of gender per age or about comorbidities per age and gender or the assumption of independence between risk factors are expected to have a small impact on results. A final point of paramount importance is that the equation presented here can be used to determine the probability of dying from COVID-19 for a particular patient, given its age interval, gender and comorbidities associated.


title: A perspective of viruses and the outbreak of a novel coronavirus SARS-CoV-2

text: The article presents some introductory remarks on viruses and in general how they lead to diseases. With reference to same, it discusses the emergence of a fairly novel coronavirus, 2019-CoV-2 and its possible medication.


Oncology care in the era of pandemics


title: Wuhan novel coronavirus 2019nCoV

text: Information about the open-access article 'Wuhan novel coronavirus 2019nCoV' in DOAJ DOAJ is an online directory that indexes and provides access to quality open access, peer-reviewed journals


title: SARS-CoV-2 infection in Health Care Workers in a large public hospital in Madrid, Spain, during March 2020

text: Background On January 31st the first case of COVID-19 was detected in Spain, an imported case from Germany in Canary Islands, and thereafter on February 25th the first case was detected in Madrid. The first case of COVID-19 was confirmed at the Hospital Universitario 12 de Octubre on March 1st, a large public hospital with 1200 beds, covering an area over 400000 inhabitants in southern Madrid. During March 2020 highly active circulation of SARS-CoV-2 was experienced in Madrid with 24090 cases officially reported by March 29th. Methods Since the beginning of the epidemics the Occupational Health and Safety Service (OHSS) organized the consulting and testing of the hospital personnel with confirmed exposure and also those presenting symptoms suggestive of viral respiratory infection. For molecular diagnosis of SARS-CoV-2 infection both nasopharyngeal and oropharyngeal swabs were obtained from suspected cases and processed at the Microbiology Laboratory by automatized specific PCR methods that was operative from February 25th as part of the preparedness. Results From a total of 6800 employees of the hospital, 2085 (30,6 %) were tested during the period 1-29 March 2020, some of them repeatedly (2286 total samples). The first HCW infected was confirmed on March 9th. A total of 791 HCW and personnel were confirmed to be infected by March 29th, representing 38% of those tested and 11,6 % of all the hospital workers. The proportion of infected individuals was estimated among the different groups of occupational exposure and the evolution of the cases during the expansive epidemic wave was compared between HCW and those patients attending at the Emergency Department (ER) during the same period and adjusted by the same age range. There were no statistically significant differences in the proportion of SARS-CoV-2 positive PCR detection between HCW from high risk areas involved in close contact with COVID-19 patients in comparison with clerical, administrative or laboratory personnel without direct contact with patients. The curves of evolution of accumulated cases between patients and HCW during March 2020 showed an almost parallel shape. Discussion The recommendation from our OHSS did not include testing of asymptomatic cases but was highly proactive in testing even patients with minor symptoms therefore, a high proportion of HCW and non-sanitary personnel was tested in March 2020 during the rapid period of expansion of the epidemics in Madrid, accounting for a total of 30,6 % of the hospital employees. Most of the COVID-19 cases among the hospital HCW and personnel were mild and managed at home under self-isolation measures, however 23 (3%) required hospitalization mostly due to severe bilateral interstitial pneumonia, two of those cases required mechanical ventilation at the ICU. No fatalities occurred during the study period. Although there were some cases of highly probable transmission from COVID-19 patients to HCWs, mainly at the first phase of the epidemics, there were no significant differences on the infection rates of HCW and hospital personnel that can be related to working in areas of high exposure risk. Furthermore, the evolution of cases during the same time period (March 2020) between patients attending the ER and hospital staff suggests that both groups were driven by the same dynamics. This experience is similar to the communicated from Wuhan verified by the WHO Joint Mission and also from recent experiences at hospital in the Netherlands, where most of the infections of HCW were related to household or community contacts. Significance Since the collective of hospital HCW are exhaustively screened in specific centers, their rate of infection for SARS-CoV-2 could be an indicator of the epidemic dynamics in the community. There appears to be a close connection between HCW infection and the driving forces of transmission in the community. Although we cannot exclude an additional risk factor of infection by SARS-CoV-2 due to the fact of the hospital environment, the similar proportions of positive cases among all the areas of the hospital and the evolutive wave of infection, as compared with the community, are clear arguments against a major factor of occupational risk. Exhaustive testing, such as the one carried out in our institution, covering over one third of all the workers, could be used as a reference of the population infected in the community. Since a significant proportion of COVID-19 cases can be asymptomatic and not all the hospital employees were actually tested, it is highly likely that this 11,6 % is a minimum estimation of the impact of SARS-CoV-2 circulation in Madrid during the first 4 weeks of the epidemics. This is in high and clear contrast with the official figures circulating at national and international levels. This has important implications to more precisely estimate the actual number of cases in the community and to develop public health policies for containment, treatment and recovery.


The Unique Challenges and Lessons Imparted by the Cystic Fibrosis Community in the Time of COVID-19 Pandemic


title: The ongoing COVID-19 epidemic in Minas Gerais, Brazil: insights from epidemiological data and SARS-CoV-2 whole genome sequencing.

text: The recent emergence of a previously unknown coronavirus (SARS-CoV-2), first confirmed in the city of Wuhan in China in December 2019, has caused serious public health and economic issues due to its rapid dissemination worldwide. Although 61,888 confirmed cases had been reported in Brazil by 28 April 2020, little was known about the SARS-CoV-2 epidemic in the country. To better understand the recent epidemic in the second most populous state in southeast Brazil (Minas Gerais, MG), we looked at existing epidemiological data from 3 states and sequenced 40 complete genomes from MG cases using Nanopore. We found evidence of multiple independent introductions from outside MG, both from genome analyses and the overly dispersed distribution of reported cases and deaths. Epidemiological estimates of the reproductive number using different data sources and theoretical assumptions all suggest a reduction in transmission potential since the first reported case, but potential for sustained transmission in the near future. The estimated date of introduction in Brazil was consistent with epidemiological data from the first case of a returning-traveler from Lombardia, Italy. These findings highlight the unique reality of MGs epidemic and reinforce the need for real-time and continued genomic surveillance strategies as a way of understanding and therefore preparing against the epidemic spread of emerging viral pathogens.


title: Lectin-like Intestinal Defensin Inhibits 2019-nCoV Spike binding to ACE2

text: The burgeoning epidemic caused by novel coronavirus 2019 (2019-nCoV) is currently a global concern. Angiotensin-converting enzyme-2 (ACE2) is a receptor of 2019-nCoV spike 1 protein (S1) and mediates viral entry into host cells. Despite the abundance of ACE2 in small intestine, few digestive symptoms are observed in patients infected by 2019-nCoV. Herein, we investigated the interactions between ACE2 and human defensins (HDs) specifically secreted by intestinal Paneth cells. The lectin-like HD5, rather than HD6, bound ACE2 with a high affinity of 39.3 nM and weakened the subsequent recruitment of 2019-nCoV S1. The cloak of HD5 on the ligand-binding domain of ACE2 was confirmed by molecular dynamic simulation. A remarkable dose-dependent preventive effect of HD5 on 2019-nCoV S1 binding to intestinal epithelial cells was further evidenced by in vitro experiments. Our findings unmasked the innate defense function of lectin-like intestinal defensin against 2019-nCoV, which may provide new insights into the prevention and treatment of 2019-nCoV infection.


title: Study of the Sudanese perceptions of COVID-19: Applying the Health Belief Model.

text: Abstract Background COVID-19 a pandemic declared by WHO is the first in recent history pose challenges on public health. Health Belief Model is a psychosocial model explains and predicts health-related behaviours. This study aimed to explore the perceptions of the Sudanese on COVID-19-related preventive measures. Methods A Cross-sectional study using online-questionnaire was conducted between 1st-16th April 2020 among Sudanese adults. We used a snowball sampling technique starting from known professional and social media groups and individuals and then was distributed on various internet platforms. The survey instrument was based on HBM constructs. Results Some 877 individuals participated in the survey with a mean age of 37.8 more males mostly having a university education employed and residing in Khartoum. More than half of the participants scored high in almost all Health Belief Model constructs except for benefits of hand hygiene. The findings show that the HBM constructs are correlated to each other as well as to other socio-demographic factors. Self-efficacy correlated negatively with susceptibility while positively with severity benefits of and barriers to hand hygiene, benefits and barriers to social distancing respectively. Conclusion The findings show that the HBM constructs are correlated to each other as well as to other socio-demographic factors. Self-efficacy must be taken into account as a strong changing factor to susceptibility and severity perceptions. Correlations found in this study might help drive behaviour-changing efforts.


title: J&J picks partners for COVID-19 vaccine

text: Johnson & Johnson has hired Emergent BioSolutions as a US manufacturing partner for its lead COVID-19 vaccine candidate Under the pact, worth about $135 million, Emergent will make the vaccine’s active ingredient starting later this year at its facility in Baltimore J&J also signed a deal to use capacity at Catalent’s biologic drug facility in Bloomington, Indiana J&J has a plan to produce 1 billion doses of its adenovirus-based vaccine View: PDF ;Full Text HTML


title: Reciprocal association between participation to a national election and the epidemic spread of COVID-19 in France: nationwide observational and dynamic modeling study.

text: Objective: To investigate possible reciprocal associations between the intensity of the COVID-19 epidemic in France and the level of participation at national elections. Design: Observational study and dynamic modelling using a sigmoidal mixed effects model. Setting: All hospitals where patients were admitted for COVID-19. Participants: All admitted patients from March 18, 2020 to April 17, 2020. Main outcome measures: Abstention and admission rate for COVID-19. Results: Mean abstention rate in 2020 among departments was 52.5%+/-6.4 and had increased by a mean of 18.8% as compared with the 2014 election. There was a high degree of similarity of abstention between the two elections among the departments (p<0.001). Among departments with a high outbreak intensity before the election, those with a higher participation were not affected by a significantly higher number of COVID-19 admissions after the elections. The sigmoidal model fitted the data from the different departments with a high degree of consistency. The covariate analysis showed that a significant association between participation and number of admitted patients was observed for both elections (2020: B=-5.36, p<1e-9 and 2014: B=-3.15, p<1e-6) contradicting a direct specific causation of the 2020 election. Participation was not associated with the position of the inflexion point suggesting no effect in the speed of spread. Conclusions: Our results suggest that the surrounding intensity of the COVID-19 epidemic in France did not have any local impact on citizens participation to a national election. The level of participation to the 2020 election had no impact on the spread of the pandemic.


title: Objective olfactory testing in patients presenting with sudden onset olfactory dysfunction as the first manifestation of confirmed COVID-19 infection

text: Abstract Background: The aims of this study are to investigate the COVID-19 status of patients with initial sudden olfactory anosmia (ISOA) using nasopharyngeal swabs for RT-PCR analysis and to explore their olfactory dysfunctions with psychophysical olfactory evaluation. Methodology: This prospective study included 78 ISOA patients who fulfilled a patient-reported outcome questionnaire and underwent a nasopharyngeal swabs. Among these, 46 patients performed psychophysical olfactory evaluation using sniffing tests. Based on the duration of the ISOA, two groups of patients were compared: patients with anosmia duration [≤]12 days (group 1) and those with duration >12 days (group 2). Results: Among group 1, 42 patients (87.5%) had a positive viral load regarding RT-PCR while 6 patients (12.5%) were negative. In group 2, 7 patients (23%) had a positive viral load and 23 patients (77%) were negative. Among the 46 patients having performed a psychophysical olfactory evaluation, we observed anosmia in 52% (N=24), hyposmia in 24% (N=11) and normosmia in 24% (N=11) of patients. The viral load significantly decreased throughout the 14-days following the onset of the olfactory disorder. Conclusions: Our results support that a high proportion of ISOA patients are Covid+. Our study supports the need to add anosmia to the list of symptoms used in screening tools for possible COVID-19 infection.


title: Relationship between odor intensity estimates and COVID-19 population prediction in a Swedish sample

text: In response to the COVID-19 pandemic, countries have implemented various strategies to reduce and slow the spread of the disease in the general population. For countries that have implemented restrictions on its population in a step-wise manner, monitoring of COVID-19 prevalence is of importance to guide decision on when to impose new, or when to abolish old, restrictions. We are here determining whether measures of odor intensity in a large sample can serve as one such measure. Online measures of how intense common household odors are perceived and symptoms of COVID-19 were collected from 2440 Swedes. Average odor intensity ratings were then compared to predicted COVID-19 population prevalence over time in the Swedish population and were found to closely track each other (r=-0.83). Moreover, we found that there was a large difference in rated intensity between individuals with and without COVID-19 symptoms and number of symptoms was related to odor intensity ratings. Finally, we found that individuals progressing from reporting no symptoms to subsequently reporting COVID-19 symptoms demonstrated a large drop in olfactory performance. These data suggest that measures of odor intensity, if obtained in a large and representative sample, can be used as an indicator of COVID-19 disease in the general population. Importantly, this simple measure could easily be implemented in countries without widespread access to COVID-19 testing or implemented as a fast early response before wide-spread testing can be facilitated.


docid: ufpdxxtk

title: An Analytic Platform for the Rapid and Reproducible Annotation of Ventilator Waveform Data

text: Algorithmic classifiers are crucial components of clinical decision support (CDS) systems needed to advance healthcare delivery. Robust CDS systems must be derived and validated via creation of multi-reviewer adjudicated gold standard datasets. Manual annotation of physiologic data such as mechanical ventilator waveform data (VWD) can be time-consuming, and lacks methodological consistency in dataset development. To address these issues, we have created a system for annotating and adjudicating VWD called the Annotation PipeLine (APL) to optimize VWD annotation by expert reviewers. APL combines visual assessment of waveform characteristics with metadata display, enabling inclusion of quantitative thresholds into annotation decisions by reviewers. APL also includes specific features for resolving multi-reviewer disagreements and generating gold standard data sets. APL’s unique combination of methods and open source framework may accelerate the creation of CDS algorithms for ventilator management, and may serve as a model for future research into physiologic waveform annotation systems.


docid: jtsxpjhc

title: Clinical features and laboratory inspection of novel coronavirus pneumonia (COVID-19) in Xiangyang, Hubei

text: Background: Since December 2019, a novel coronavirus pneumonia (COVID-19) rapidly spread in China, reached multiple continents currently.We aimed to reveal the infectious characteristics of COVID-19 that provide more information for the research of novel coronavirus. Methods: We performed a retrospective study on the clinical characteristics of 128 COVID-19 cases with laboratory-confirmed from Xiangyang No 1 Hospitalad during January 2020 to 16 February 2020. Results: Female patients account for 53.1%. The aged below 20 years that accounts for 1.6% of overall patients. The aged in 21~50, 51~65, over 66 years were accounts for 44.5%, 35.1%,18.8%, respectively. In the difference age spectrum, all severe groups compared with non-severe groups were difference significantly ( P < 0.01 ). Fever ( 89.8% ) and Cough ( 67.2% ) were common clinical symptoms. The rate of patients with sore throats (14.1%) was rare. The rate of chest computed tomography scan showing ground glass opacity in overall, non-severe, severe groups were 63.3%, 60.7%, 76.2%, respectively. White blood cell counts in the normal range of overall patients, but severe group patients were increased significantly ( P < 0.01). Lymphocytes of overall patients were decreased. Alanine transaminase (ALT) and aspartate transaminase (AST) in the normal range of overall patients, but its were elevated in the severe group. Creatinine (CR) and blood urea nitrogen (BUN) of overall patients in the normal range. C-reactive protein (CRP) level of all patients were increased markedly, but it in the severe group was significantly higher than that in the non-severe group ( P < 0.01 ). Conclusions: Our data provide more information that advanced age, lower lymphocytes levels at the diagnosed COVID-19 patients may be a risk factor for unfavourable prognosis. The white blood cells and C-reactive protein level elevated in severe COVID-19 patients may be accompanying bacterial infection. 2019-nCov may be carries a risk factor of impaired liver and kidney function.


title: A pilot study to investigate the fecal dissemination of SARS-CoV-2 virus genome in COVID-19 patients in Odisha, India

text: In infectious diseases, the routes of transmission play major roles in determining the rate and extent of disease spread. Though fomites and aerosol droplets are major sources of SARS-CoV-2 human to human transmission, studies have also reported possible involvement of other routes of transmission like fecal-oral. Multiple studies around the world have reported shedding of the SARS-CoV-2 viral genome in certain COVID-19 patient fecal samples. Hence, the major objective of this study was to get the experimental evidence whether in Indian COVID-19 patients fecal dissemination of the SARS-CoV-2 genome occurs or not. Information obtained from twelve number of patients from a COVID-19 hospital of Odisha has demonstrated that both symptomatic and asymptomatic Indian patients could be positive for the SARS-CoV-2 genome in their fecal component. The findings have also established a protocol to collect and extract viral RNA for SARS-CoV-2 detection in fecal samples. Together, the study supports the hypothesis of possible fecal-oral transmission of SARS-CoV-2 virus and provides a rationale to extend this study in a larger cohort of patient samples and correlate the significance of the SARS-CoV-2 virus genome detection in fecal samples with disease severity and transmission.


title: Estimating Probabilities of Success of Clinical Trials for Vaccines and Other Anti-Infective Therapeutics

text: A key driver in biopharmaceutical investment decisions is the probability of success of a drug development program. We estimate the probabilities of success (PoS) of clinical trials for vaccines and other anti-infective therapeutics using 43,414 unique triplets of clinical trial, drug, and disease between January 1, 2000, and January 7, 2020, yielding 2,544 vaccine programs and 6,829 non-vaccine programs targeting infectious diseases. The overall estimated PoS for an industry-sponsored vaccine program is 39.6%, and 16.3% for an industry-sponsored anti-infective therapeutic. Among industry-sponsored vaccines programs, only 12 out of 27 disease categories have seen at least one approval, with the most successful being against monkeypox (100%), rotavirus (78.7%), and Japanese encephalitis (67.6%). The three infectious diseases with the highest PoS for industry-sponsored non-vaccine therapeutics are smallpox (100%), CMV (31.8%), and onychomycosis (29.8%). Non-industry-sponsored vaccine and non-vaccine development programs have lower overall PoSs: 6.8% and 8.2%, respectively. Viruses involved in recent outbreaks---MERS, SARS, Ebola, Zika---have had a combined total of only 45 non-vaccine development programs initiated over the past two decades, and no approved therapy to date (Note: our data was obtained just before the COVID-19 outbreak and do not contain information about the programs targeting this disease.) These estimates offer guidance both to biopharma investors as well as to policymakers seeking to identify areas most likely to be undeserved by private-sector engagement and in need of public-sector support.


title: The QT Interval in Patients with SARS-CoV-2 Infection Treated with Hydroxychloroquine/Azithromycin

text: We report the change in the QT interval in 84 adult patients with SARS-CoV-2 infection treated with Hydroxychloroquine/Azithromycin combination. QTc prolonged maximally from baseline between days 3 and 4. in 30% of patients QTc increased by greater than 40ms. In 11% of patients QTc increased to >500 ms, representing high risk group for arrhythmia. The development of acute renal failure but not baseline QTc was a strong predictor of extreme QTc prolongation.


title: Genetic variants that influence SARS-CoV-2 receptor TMPRSS2 expression among population cohorts from multiple continents

text: The World Health Organization recently announced that pandemic status has been achieved for coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exponential increases in patient numbers have been reported around the world, along with proportional increases in the number of COVID-19-related deaths. The SARS-CoV-2 infection rate in a population is expected to be influenced by social practices, availability of vaccines or prophylactics, and the prevalence of susceptibility genes in the population. Previous work revealed that cellular uptake of SARS-CoV-2 requires Angiotensin Converting Enzyme 2 (ACE-2) and a cellular protease. The spike (S) protein on SARS-CoV-2 binds ACE-2, which functions as an entry receptor. Following receptor binding, transmembrane protease serine 2 (encoded by TMPRSS2) primes the S protein to allow cellular uptake. Therefore, individual expression of TMPRSS2 may be a crucial determinant of SARS-CoV-2 infection susceptibility. Here, we utilized multiple large genome databases, including the GTEx portal, SNP nexus, and Ensembl genome project, to identify gene expression profiles for TMPRSS2 and its important expression quantitative trait loci. Our results show that four variants (rs464397, rs469390, rs2070788 and rs383510) affect expression of TMPRSS2 in lung tissue. The allele frequency of each variant was then assessed in regional populations, including African, American, European, and three Asian cohorts (China, Japan and Taiwan). Interestingly, our data shows that TMPRSS2-upregulating variants are at higher frequencies in European and American populations than in the Asian populations, which implies that these populations might be relatively susceptible to SARS-CoV-2 infection.


title: WHOLE-GENOME SEQUENCING AND DE NOVO ASSEMBLY OF A 2019 NOVEL CORONAVIRUS (SARS-COV-2) STRAIN ISOLATED IN VIETNAM

text: The pandemic COVID-19 caused by the zoonotic virus SARS-CoV-2 has devastated countries worldwide, infecting more than 4.5 million people and leading to more than 300,000 deaths. Whole genome sequencing (WGS) is an effective tool to monitor emerging strains and provide information for intervention, thus help to inform outbreak control decisions. Here, we reported the first effort to sequence and de novo assemble the whole genome of SARS-CoV-2 using PacBio’s SMRT sequencing technology in Vietnam. We also presented the annotation results and a brief analysis of the variants found in our SARS-CoV-2 strain, which was isolated from a Vietnamese patient. The sequencing was successfully completed and de novo assembled in less than 30 hours, resulting in one contig with no gap and a length of 29,766 bp. All detected variants as compared to the NCBI reference were highly accurate as confirmed by Sanger sequencing. The results have shown the potential of long read sequencing to provide high quality WGS data to support public health responses, and advance understanding of this and future pandemics.


title: Psychological responses, behavioral changes and public perceptions during the early phase of the COVID-19 outbreak in China: a population based cross-sectional survey

text: Objective: To investigate psychological and behavioral responses to the threat of SARS-CoV-2 infections and their associations with public perceptions in China Design: Cross sectional population-based telephone survey via random digital dialing between 1 and 10 February, 2020 Setting: Wuhan (the epicentre and quarantined city), and Shanghai (a typical major city with close transportation link with Wuhan) Participants: Random sample of 510 residents in Wuhan and 501 residents in Shanghai aged above 18 Main outcome measures: Anxiety (measured by the 7-item generalized anxiety disorder [GAD-7] scale), recommended and avoidance behaviors (engaged in all six behaviors such as increasing surface cleaning and reducing going out). Results: The prevalence rates of moderate or severe anxiety (score ≥10 on GAD-7) were 32.7% (n=167) among Wuhan participants and 20.4% (n=102) among Shanghai participants. 78.6% (n=401) of Wuhan participants and 63.9% (n=320) of Shanghai participants had carried out all six precautionary behaviors. For both measures, Wuhan participants were more responsive to the outbreak (p<0.001). Controlling for personal characteristics, logistic regression results suggested that risks of moderate or severe anxiety were positively associated with perceived susceptibility (odds ratio 1.6, 95% confidence interval 1.3-1.8) and severity of the disease (1.6, 1.4-1.9) and confusion about information reliability (1.6, 1.5-1.9). Having confidence in taking measures to protect oneself against the disease was associated with a lower risk (0.6, 0.5-0.7). The strongest predictor of behavioral change was perceived severity (1.2, 1.1-1.4), followed by confusion about information reliability (1.1, 1.0-1.3). Conclusions: Psychological and behavioral responses to COVID-19 have been dramatic during the rising phase of the outbreak. Our results support efforts for timely dissemination of accurate and reliable information to address the high anxiety level.


title: The Challenge of Teaching Mission in an Increasingly Mobile and Complex World

text: In a time when old groupings are breaking up, when people are on the move or fleeing conflict and settling elsewhere, when many people are multilingual and are competent in several cultural settings, doing mission has become an ever greater challenge than simple cross-cultural communication. How does one engage in mission on the run? How does one conduct missiological research in ephemeral community that is here today and gone tomorrow? How does one train new missionaries for work in a field that will certainly morph into something else before they get there? Changing times call for new perspectives on mission.


title: Gender differences in patients with COVID-19: Focus on severity and mortality

text: Importance: The recent outbreak of Novel Coronavirus (SARS-CoV-2) Disease (COVID-19) has put the world on alert, that is reminiscent of the SARS outbreak seventeen years ago. Objective: We aim to compare the severity and mortality between male and female patients with both COVID-19 and SARS, to explore the most useful prognostic factors for individualized assessment. Design, Setting, and Participants: We extracted the data from a case series of 43 hospitalized patients we treated, a public data set of the first 37 cases died of COVID-19 in Wuhan city and 1019 survived patients from six cities in China. We also analyzed the data of 524 patients with SARS, including 139 deaths, from Beijing city in early 2003. Main Outcomes and Measures: Severity and mortality. Results: Older age and high number of comorbidities were associated with higher severity and mortality in patients with both COVID-19 and SARS. The percentages of older age (≥65 years) were much higher in the deceased group than in the survived group in patients with both COVID-19 (83.8 vs. 13.2, P<0.001) and SARS (37.4 vs. 4.9, P<0.001). In the case series, men tend to be more serious than women (P=0.035), although age was comparable between men and women. In the public data set, age was also comparable between men and women in the deceased group or the survived group in patients with COVID-19. Meanwhile, gender distribution was exactly symmetrical in the 1019 survivors of COVID-19. However, the percentage of male were higher in the deceased group than in the survived group (70.3 vs. 50.0, P=0.015). The gender role in mortality was also observed in SARS patients. Survival analysis showed that men (hazard ratio [95% CI] 1.47 [1.05-2.06, P= 0.025) had a significantly higher mortality rate than women in patients with SARS. Conclusions and Relevance: Older age and male gender are risk factors for worse outcome in patients with COVID. While men and women have the same susceptibility to both SARS-CoV-2 and SARS-CoV, men may be more prone to have higher severity and mortality independent of age and susceptibility.


title: Vibration Assisted AFM-Based Nanomachining under Elevated Temperatures using Soft and Stiff Probes

text: Abstract Atomic force microscope (AFM)-based nanomanufacturing, as a low-cost and easy-to-setup technique, enables high-resolution maskless nanofabrication processes for arbitrary nanopatterns, but there are strong needs to advance the nanopatterning processes. Thermal-mechanical AFM-based nanomachining process lower the normal forces to few hundreds of nanonewtons, but it requires special thermal probes. Vibration-assisted nanomachining process increases the tip lifetime but forces are still in the range of few hundreds of nanonewtons. We have innovated a system with both vibration and heated sample to achieve nanomachining process with lower normal forces. To provide cantilever selection guidance for this novel nanomachining system and to understand the effect of cantilever stiffness and sample temperature on feature dimensions, we used both soft and stiff probes for the nanomachining processes in this paper. We fabricated nanostructures with controllable shape dimensions were fabricated on 200 nm depth polymethyl methacrylate (PMMA) films by using both soft and stiff AFM probes with a few tens of nanonewtons normal forces in this novel AFM-based nanomachining system. Besides, we analyzed the effects of different machining parameters on the lithography performance, which helps to understand the machining parameters for fabricating nanopatterns of different dimensions. We also found that other than the setpoint force, the probe category and sample heating temperature significantly affect the dimensions of fabricated nanopatterns. And there is a statistically significant interaction effect between the spring constants of probes and the sample temperatures.


title: Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

text: The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses −1 programmed ribosomal frameshifting (−1 PRF) to control the relative expression of viral proteins. As modulating −1 PRF can inhibit viral replication, the RNA pseudoknot stimulating −1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by μs-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting −1 PRF in SARS-CoV-2.


title: Diagnosis and treatment strategies for acute biliary infection during the epidemic period of COVID-19/ 新型冠状病毒肺炎疫情期间急性胆道感染的诊疗策略

text: Acute biliary infection is one of the common causes of acute abdomen, easily causes severe infection and even death. The reasonable treatment of the acute phase is very important. The sudden outbreak of corona virus disease (COVID-19) has posed severe challenges to the country's economic and social life, and has also led to an extreme shortage of medical resources. The diagnosis and treatment of acute biliary infections disease also face challenges. In order to control the epidemic of infectious diseases, we write this article from the perspective of prevention and controlling of COVID-19, referring to the Tokyo Guidelines 2018 to express our views on the diagnosis and treatment strategies for acute biliary infection, hoping to do our best to prevent and control the epidemic of COVID-19.


COVID-19 Infection and Circulating ACE2 Levels: Protective Role in Women and Children


title: Reopening universities during the COVID-19 pandemic: A testing strategy to minimize active cases and delay outbreaks

text: Background: University campuses present an ideal environment for viral spread and are therefore at extreme risk of serving as a hotbed for a COVID-19 outbreak. While active surveillance throughout the semester such as widespread testing, contact tracing, and case isolation, may assist in detecting and preventing early outbreaks, these strategies will not be sufficient should a larger outbreak occur. It is therefore necessary to limit the initial number of active cases at the start of the semester. We examine the impact of pre-semester NAT testing on disease spread in a university setting. Methods: We implement simple dynamic transmission models of SARS-CoV-2 infection to explore the effects of pre-semester testing strategies on the number of active infections and occupied isolation beds throughout the semester. We assume an infectious period of 3 days and vary R0 to represent the effectiveness of disease mitigation strategies throughout the semester. We assume the prevalence of active cases at the beginning of the semester is 5%. The sensitivity of the NAT test is set at 90%. Results: If no pre-semester screening is mandated, the peak number of active infections occurs in under 10 days and the size of the peak is substantial, ranging from 5,000 active infections when effective mitigation strategies (R0 = 1.25) are implemented to over 15,000 active infections for less effective strategies (R0 = 3). When one NAT test is mandated within one week of campus arrival, effective (R0 = 1.25) and less effective (R0 = 3) mitigation strategies delay the onset of the peak to 40 days and 17 days, respectively, and result in peak size ranging from 1,000 to over 15,000 active infections. When two NAT tests are mandated, effective (R0 = 1.25) and less effective (R0 = 3) mitigation strategies delay the onset of the peak through the end of fall semester and 20 days, respectively, and result in peak size ranging from less than 1,000 to over 15,000 active infections. If maximum occupancy of isolation beds is set to 2% of the student population, then isolation beds would only be available for a range of 1 in 2 confirmed cases (R0 = 1.25) to 1 in 40 confirmed cases (R0 = 3) before maximum occupancy is reached. Conclusion: Even with highly effective mitigation strategies throughout the semester, inadequate pre-semester testing will lead to early and large surges of the disease and result in universities quickly reaching their isolation bed capacity. We therefore recommend NAT testing within one week of campus return. While this strategy is sufficient for delaying the timing of the outbreak, pre-semester testing would need to be implemented in conjunction with effective mitigation strategies to reduce the outbreak size.


title: Sleep quality, mental health and circadian rhythms during COVID lockdown: Results from the SleepQuest Study

text: Behavioural responses to COVID19 lockdown will define the long-term impact of psychological stressors on sleep and brain health. Here we tease apart factors that help protect against sleep disturbance. We capitalise on the unique restrictions during COVID19 to understand how time of day of daylight exposure and outside exercise interact with chronotype and sleep quality. 3474 people from the UK (median age 62, range 18 to 91) completed our online 'SleepQuest' Study between 29th April and 13th May 2020 - a set of validated questionnaires probing sleep quality, depression, anxiety and attitudes to sleep alongside bespoke questions on the effect of COVID19 lockdown on sleep, time spent outside and exercising and self-help sleep measures. Significantly more people (n=1252) reported worsened than improved sleep (n=562) during lockdown (p<0.0001). Factors significantly associated with worsened sleep included low mood (p<0.001), anxiety (p<0.001) and suspected, proven or at risk of COVID19 symptoms (all p<0.03). Sleep improvement was related to the increased length of time spent outside (P<0.01). Older people's sleep quality was less affected than younger people by COVID19 lockdown (p<0.001). Better sleep quality was associated with going outside and exercising earlier, rather than later, in the day. However, the benefit of being outside early is driven by improved sleep in 'owl' (p=0.0002) and not 'lark' (p=0.27) chronotype, whereas, the benefit of early exercise (inside or outside) did not depend on chronotype. Defining the interaction between chronotype, mental health and behaviour will be critical for targeted lifestyle adaptations to protect brain health through current and future crises.


title: Impact of changing case definitions for COVID-19 on the epidemic curve and transmission parameters in mainland China

text: Background: When a new infectious disease emerges, appropriate case definitions are important for clinical diagnosis and also for public health surveillance. Tracking case numbers over time allows us to determine speed of spread and the effectiveness of interventions. Changing case definitions during an epidemic can affect these inferences. Methods: We examined changes in the case definition for COVID-19 in mainland China during the first epidemic wave. We used simple models assuming exponential growth and then exponential decay to estimate how changes in the case definitions affected the numbers of cases reported each day. We then inferred how the epidemic curve would have appeared if the same case definition had been used throughout the epidemic. Findings: From January through to early March 2020, seven versions of the case definition for COVID-19 were issued by the National Health Commission in China. As of February 20, there were 55,508 confirmed cases reported in mainland China. We estimated that when the case definitions were changed from version 1 to 2, version 2 to 4 and version 4 to 5, the proportion of infections being detected as cases were increased by 7.1-fold (95% credible interval (CI): 4.8, 10.9), 2.8-fold (95% CI: 1.9, 4.2) and 4.2-fold (95% CI: 2.6, 7.3) respectively. If the fifth version of the case definition had been applied throughout the outbreak, we estimated that by February 20 there would have been 232,000 (95% CI: 161,000, 359,000) confirmed cases. Interpretation: The case definition was initially narrow, but was gradually broadened to allow detection of more cases as knowledge increased, particularly milder cases and those without epidemiological links to Wuhan or other known cases. This should be taken into account when making inferences on epidemic growth rates and doubling times, and therefore on the reproductive number, to avoid bias. Funding: Commissioned grant from the Health and Medical Research Fund, Food and Health Bureau, Government of the Hong Kong Special Administrative Region.


title: Spousal bereavement, mortality and risk of negative health outcomes among older adults: a population-based study

text: Objective: to examine whether spousal bereavement increases the risk of death and negative health outcomes and among older people. Design: cohort study and self-controlled cohort crossover study Setting: routinely collected administrative and healthcare data with individual-level linkage between several national registries in Sweden. Participants: older persons (>65 years) living in the community whose spouse died in 2013-2014, individually matched with controls. Main outcome measures: death from any cause (primary outcome), acute cardiovascular events, pneumonia, hip fracture, and intentional self-harm (secondary outcomes). In the cohort study, incidence rate ratios were estimated with conditional fixed-effect Poisson regression models adjusted for potential confounders. In the self-controlled cohort crossover study, relative incidence ratios were estimated over the 12 months before and after spousal loss with unadjusted conditional fixed-effect Poisson regression including a bereavement-by-time interaction. Results: 42 918 bereaved older spouses were included and matched to an equal number of married controls (mean age 78.9 [SD 7.2] years, 68% women). During the first year of follow-up, the risk of death from any cause was 1.66 (95% confidence interval 1.53 to 1.80) times higher for bereaved cases than for married controls, and bereaved cases survived on average 4.2 days shorter than married controls. Bereaved cases also experienced an increased risk of acute cardiovascular events (incidence rate ratio 1.34, 1.24 to 1.44), hip fracture (1.48, 1.30 to 1.68), pneumonia (1.14, 1.04 to 1.25), and self-harm (3.49, 2.11 to 5.76). These associations were strongly time-dependent, increasing sharply immediately after spousal loss and weakening as time elapsed. In the self-controlled cohort crossover study, the relative incidence ratios increased for all four secondary outcomes, starting already during the 6-month period preceding spousal loss. Conclusion: Among older persons, the association between spousal bereavement and the risk of negative health outcomes and mortality is most likely causal. Our finding that the risk of adverse health consequences increases already during the 6 months prior to spousal loss indicates that palliative care services have an important role to play in providing timely bereavement care to spouses and other family caregivers.


title: Scalable, rapid and highly sensitive isothermal detection of SARS-CoV-2 for laboratory and home testing

text: Global efforts to monitor and contain the Covid-19 pandemic, caused by the beta-coronavirus SARS-CoV-2, currently rely on RT-qPCR-based diagnostic assays. Yet their high cost, moderate throughput, and dependence on sophisticated equipment limit a broad implementation. Loop-mediated isothermal amplification (RT-LAMP) is an alternative detection method that has the potential to overcome these limitations. Here, we established a robust, highly sensitive and versatile RT-LAMP-based SARS-CoV-2 detection assay that is insensitive to carry-over contaminations. Our approach uses a rapid upfront lysis step and hydroxy-naphthol-blue (HNB) for colorimetric detection, which enables the robust identification of Covid-19 infections from a variety of sample types within 30 minutes. By combining RT-LAMP with a simple nucleic acid enrichment method (bead-LAMP), we profoundly increased assay sensitivity to RT-qPCR-like levels, thereby extending applications to large-scale pooled testing. Finally, we developed HomeDip-LAMP for pipette-free SARS-CoV-2 detection for low-resource environments. Our combined optimizations set the stage for implementing RT-LAMP as SARS-CoV-2 diagnostics assay for population-wide and home-based testing.


title: Convalescent plasma therapy for the treatment of patients with COVID-19: Assessment of methods available for antibody detection and their correlation with neutralising antibody levels

text: Introduction. The lack of approved specific therapeutic agents to treat COVID-19 associated with SARS coronavirus 2 (SARS-CoV-2) infection has led to the rapid implementation and/or randomised controlled trials of convalescent plasma therapy (CPT) in many countries including the UK. Effective CPT is likely to require high titres of neutralising antibody levels in convalescent donations. Understanding the relationship between functional neutralising antibodies and antibody levels to specific SARS-CoV-2 proteins in scalable assays will be crucial for the success of large-scale collection and use of convalescent plasma. We assessed whether neutralising antibody titres correlated with reactivity in a range of ELISA assays targeting the spike (S) protein, the main target for human immune response. Methods. Blood samples were collected from 52 individuals with a previous laboratory confirmed SARS-CoV-2 infection at least 28 days after symptom resolution. These were assayed for SARS-CoV-2 neutralising antibodies by microneutralisation and pseudotype assays, and for antibodies by four different ELISAs. ROC analysis was used to further identify sensitivity and specificity of selected assays to identify samples containing high neutralising antibody levels suitable for clinical use of convalescent plasma. Results. All samples contained SARS-CoV-2 antibodies, whereas neutralising antibody titres of greater than 1:20 were detected in 43 samples (83% of those tested) and >1:100 in 22 samples (42%). The best correlations were observed with EUROimmun IgG ELISA S/CO reactivity (Spearman Rho correlation co-efficient 0.88; p<0.001). Based on ROC analysis, EUROimmun would detect 60% of samples with titres of >1:100 with 100% specificity using a reactivity index of 9.1 (13/22). Discussion. Robust associations between virus neutralising antibody titres and reactivity in several ELISA-based antibody tests demonstrate their possible utility for scaled-up production of convalescent plasma containing potentially therapeutic levels of anti-SARS-CoV-2 neutralising antibodies.


title: Identification of viral etiology of acute respiratory tract infections in children and adults in Tabanan, Bali, Indonesia

text: Acute respiratory tract infection (ARTI) is the most common infectious disease in humans worldwide The morbidity and mortality rates are high, especially in developing countries from Southeast Asia and Africa While ARTI is commonly associated with viruses, there is limited data on the spectrum of viruses causing ARTI in developing countries, including Indonesia This study was based on utilizing molecular techniques targeting a panel of 11 endemic and emerging respiratory viral pathogens including zoonotic viruses in a cohort of children and adults presenting at Tabanan General Hospital, Bali, with acute respiratory illness, from January to November 2017 In total, 98 out of 200 samples (49 0 %) tested positive for viruses Our study confirmed 64 3 % viral etiology in children and 12 2 % in adults Viruses that were detected were Herpesviridae (15 0 %) followed by enteroviruses (12 0 %), influenza A virus (11 5 %), respiratory syncytial virus (8 0 %), Adenoviridae (6 5 %), human metapneumovirus (3 5 %), Paramyxoviridae (2 0 %), bocavirus (1 0 %) and Coronaviridae (0 5 %) The study sheds light on the viral spectrum of ARTI in children and adults in Tabanan, Bali, Indonesia


title: Assessment of Fabric Masks as Alternatives to Standard Surgical Masks in Terms of Particle Filtration Efficiency

text: In response to the critical shortage of medical masks resulting from the COVID-19 pandemic, large portions of the population are mobilizing to produce cloth masks using locally-sourced fabrics, however the efficacy of these masks as a means of protecting the wearer from airborne particles carrying virus is not well known. Further, existing protocols are designed for testing the fit and performance N95 respirators and tight-fitting facemasks rather than the relatively more loose-fitting surgical mask style most cloth masks follow. In this study tools and methods typically used to assess tight-fitting facemasks were modified to assess the efficacy of community-produced fabric and commercially-produced surgical masks in terms of protecting the wearer from airborne particles that may be carrying virus. Two TSI PortaCount (model 8028) instruments were operated concurrently to collect particle counts (particles/cm^3) in size range 0.02 to >1 um from ambient air and air just inside the breathing zone of the mask (1 measurement per second, evaluation period of 1 minute per test). Percent particle removal was determined for ten home-made, fabric masks of different designs, with and without filter layers, as well as three commercially-produced surgical-type masks. N95 masks were used to validate the method, and a 3M model 1826 surgical mask was used as a baseline for comparison of other masks of this style. Home-made masks worn as designed always had lower particle removal rates than the 3M masks, achieving between 38% and 96% of this baseline. As has been previously observed by Cooper et al. (1983), adding a layer of nylon stocking over the masks minimized the flow of air around the edges of the masks and improved particle filtration efficiency for all masks, including all commercial products tested. Use of a nylon stocking overlayer brought the particle filtration efficiency for five of the ten fabric masks above the 3M surgical mask baseline. This rapid testing method (<2 hours per mask design) provides a holistic evaluation of mask particle removal efficacy (material, design, and fit), and use of this method for testing a wider range of mask materials and designs will provide the public and health care providers with information needed to optimize health protection given resources at hand.


Recently published papers: inflammation, elucidation, manipulation?


title: An investigation of the role of transmembrane domains in Golgi protein retention.

text: The single transmembrane domains (TMDs) of the resident glycosylation enzymes of the Golgi apparatus are involved in preventing these proteins moving beyond the Golgi. It has been proposed that either the TMDs associate, resulting in the formation of large oligomers of Golgi enzymes, or that they mediate the lateral segregation of the enzymes between lipid microdomains. Evidence for either type of interaction has been sought by examining the retention of sialyltransferase (ST), an enzyme of the mammalian trans Golgi. No evidence could be obtained for specific interactions or 'kin recognition' between ST and other proteins of the trans Golgi. Moreover, it is shown that the previously described kin recognition between enzymes of the medial Golgi involves the lumenal portions of these proteins rather than their TMDs. To investigate further the role of the ST TMD, the effects on Golgi retention of various alterations in the TMD were examined. The addition or removal of residues showed that the efficiency of retention of ST is related to TMD length. Moreover, when a type I plasma membrane protein was expressed with a synthetic TMD of 23 leucines it appeared on the cell surface, but when the TMD was shortened to 17 leucines accumulation in the Golgi was observed. These observations are more consistent with lipid-based sorting of ST TMD, but they also allow for reconciliation with the kin recognition model which appears to act on sequences outside of the TMD.


title: Unique features of internal initiation of hepatitis C virus RNA translation.

text: The question of whether hepatitis C virus (HCV) RNA is translated by a mechanism of internal ribosome entry has been examined by testing whether insertion of HCV sequences between the two cistrons of a dicistronic mRNA promotes translation of the downstream cistron in rabbit reticulocyte lysates. Deletion analysis showed that efficient internal initiation required a segment of the HCV genome extending from about nucleotides 40-370 and that deletions from the 3'-end of this element were highly deleterious. As the authentic initiation codon for HCV polyprotein synthesis is at nucleotide 342, this demonstrates that, besides 5'-UTR sequences, a short length of HCV coding sequences is required for internal initiation. This finding was confirmed in transfection assays of BT7-H cells and was shown to be independent of the nature of the downstream reporter cistron. The strong requirement for coding sequences is in sharp contrast to internal initiation of picornavirus RNA translation. As a probable correlate with this, it was also found that the efficiency of internal initiation was only marginally compromised when the authentic initiation codon was mutated to a non-AUG codon, again in sharp contrast with the picornaviruses. The finding that coding sequences are required for internal initiation has important implications for the design of experiments to test for internal initiation of translation of cellular mRNAs.


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title: Structural and functional studies of retroviral RNA pseudoknots involved in ribosomal frameshifting: nucleotides at the junction of the two stems are important for efficient ribosomal frameshifting.

text: Ribosomal frameshifting, a translational mechanism used during retroviral replication, involves a directed change in reading frame at a specific site at a defined frequency. Such programmed frameshifting at the mouse mammary tumor virus (MMTV) gag-pro shift site requires two mRNA signals: a heptanucleotide shifty sequence and a pseudoknot structure positioned downstream. Using in vitro translation assays and enzymatic and chemical probes for RNA structure, we have defined features of the pseudoknot that promote efficient frameshifting. Heterologous RNA structures, e.g. a hairpin, a tRNA or a synthetic pseudoknot, substituted downstream of the shifty site fail to promote frameshifting, suggesting that specific features of the MMTV pseudoknot are important for function. Site-directed mutations of the MMTV pseudoknot indicate that the pseudoknot junction, including an unpaired adenine nucleotide between the two stems, provides a specific structural determinant for efficient frameshifting. Pseudoknots derived from other retroviruses (i.e. the feline immunodeficiency virus and the simian retrovirus type 1) also promote frameshifting at the MMTV gag-pro shift site, dependent on the same structure at the junction of the two stems.


title: Topology of the non-structural rotavirus receptor glycoprotein NS28 in the rough endoplasmic reticulum.

text: The rotavirus non-structural glycoprotein (NS28), the receptor for the virus core during budding into the lumen of the rough endoplasmic reticulum (RER), is 175 amino acids long and possesses an uncleaved signal sequence and two amino-terminal glycosylation sites. Utilizing one of three potential hydrophobic domains, the protein spans the membrane only once, with the glycosylated amino-terminal region oriented to the luminal side of the ER and the carboxy-terminal region to the cytoplasmic side. To localize sequences involved in translocation of NS28, we constructed a series of mutations in the coding regions for the hydrophobic domains of the protein. Mutant protein products were studied by in vitro translation and by transfection in vivo. In transfected cells, all mutant forms localize to the ER, and none are secreted. In vitro, each of the three hydrophobic domains is able to associate with microsomes. However, glycosylation and proteolysis of wild-type and mutant forms of NS28 indicates that the wild-type protein is anchored in the membrane only by the second hydrophobic domain, leaving approximately 131 residues exposed on the cytoplasmic side for receptor - ligand interaction.


title: CD4 cell surface downregulation in HIV-1 Nef transgenic mice is a consequence of intracellular sequestration.

text: The Nef gene product is a regulatory protein of HIV whose biological function is poorly understood. Nef has been thought to have a negative effect on viral replication in vitro but has been shown in studies with SIV to be necessary in the establishment of viraemia in vivo. In vitro studies in various human cell lines have shown that Nef downregulates the expression of cell surface CD4 and thus could have effects on the immune response. We have generated four transgenic mouse lines, with constructs containing two different Nef alleles under the control of CD2 regulatory elements to examine the interaction of Nef with the host immune system in vivo. In adult transgenic mice we have found marked downregulation in the level of CD4 on the surface of double positive thymocytes and a decrease in the number of CD4+ T cells in the thymus. Functional analyses have revealed a decrease in the total activation of transgenic thymocytes by anti-CD3 epsilon antibody. By specific intracellular staining of T cells in such mice we have found CD4 colocalizing with a Golgi-specific marker. These results strongly suggest a Nef mediated effect on developing CD4 thymocytes resulting from interference of Nef in the intracellular trafficking or post-translational modification of CD4.


title: Mof4-1 is an allele of the UPF1/IFS2 gene which affects both mRNA turnover and -1 ribosomal frameshifting efficiency.

text: The mof4-1 (maintenance of frame) allele in the yeast Saccharomyces cerevisiae was isolated as a chromosomal mutation that increased the efficiency of -1 ribosomal frameshifting at the L-A virus frameshift site and caused loss of M1, the satellite virus of L-A. Here, we demonstrate that strains harboring the mof4-1 allele inactivated the nonsense-mediated mRNA decay pathway. The MOF4 gene was shown to be allelic to UPF1, a gene whose product is involved in the nonsense-mediated mRNA decay pathway. Although cells harboring the mof4-1 allele of the UPF1 gene lose the M1 virus, mutations in other UPF genes involved in nonsense-mediated mRNA decay maintain the M1 virus. The mof4-1 strain is more sensitive to the aminoglycoside antibiotic paromomycin than a upf1 delta strain, and frameshifting efficiency increases in a mof4-1 strain grown in the presence of this drug. Further, the ifs1 and ifs2 alleles previously identified as mutations that enhance frameshifting were shown to be allelic to the UPF2 and UPF1 genes, respectively, and both ifs strains maintained M1. These results indicate that mof4-1 is a unique allele of the UPF1 gene and that the gene product of the mof4-1 allele affects both -1 ribosomal frameshifting and mRNA turnover.


title: Preformed cytoplasmic nucleocapsids are not necessary for alphavirus budding.

text: According to the present model for assembly of alphaviruses, e.g. Semliki Forest virus (SFV), the viral genome is first encapsidated into a nucleocapsid (NC) in cytoplasm and this is then used for budding at plasma membrane (PM). The preformed NC is thought to act as a template on which the viral envelope can be organized. In the present work we have characterized two SFV deletion mutants which did not assemble NCs in the cytoplasm but which instead appeared to form NCs at the PM simultaneously with virus budding. The deletions were introduced in a conserved 14 residue long linker peptide that joins the amino-terminal RNA-binding domain with the carboxy-terminal serine-protease domain of the capsid protein. Despite the deletions and the change in morphogenesis, wild-type (wt)-like particles were produced with almost wt efficiency. It is suggested that the NC assembly defect of the mutants is rescued through spike-capsid interactions at PM. The results show that the preassembly of NCs in the cytoplasm is not a prerequisite for alphavirus budding. The apparent similarities of the morphogenesis pathways of wt and mutant SFV with those of type D and type C retroviruses are discussed.


title: The P gene of bovine parainfluenza virus 3 expresses all three reading frames from a single mRNA editing site.

text: The P gene of bovine parainfluenza virus 3 (bPIV3) contains two downstream overlapping ORFs, called V and D. By comparison with the mRNA editing sites of other paramyxoviruses, two editing sites were predicted for bPIV3; site a to express the D protein, and site b to express the V protein. Examination of the bPIV3 mRNAs, however, indicates that site b is non-functional whereas site a operates frequently. Insertions at site a give rise to both V and D protein mRNAs, because a very broad distribution of Gs is added when insertions occur. This broad distribution is very different from the editing sites of Sendai virus or SV5, where predominantly one form of edited mRNA containing either a one or two G insertion respectively is created, to access the single overlapping ORF of these viruses. A model is proposed to explain how paramyxoviruses control the range of G insertions on that fraction of the mRNAs where insertions occur. The bPIV3 P gene is unique as far as we know, in that a sizeable portion of the gene expresses all 3 reading frames as protein. bPIV3 apparently does this from a single editing site by removing the constraints which control the number of slippage rounds which take place.


title: Sequences within and adjacent to the transmembrane segment of alpha-2,6-sialyltransferase specify Golgi retention.

text: The glycosyltransferase alpha-2,6-sialyltransferase (ST) is a Type II membrane protein localized to the Golgi apparatus. The first 44 amino acids of this protein were able to specify Golgi retention of a fused marker protein, lysozyme. This section of ST contains a transmembrane segment which serves as a non-cleaved signal anchor. When lysozyme was fused to an equivalent region of a cell surface protein it now appeared on the cell surface. Analysis of chimeras between the two proteins revealed that the transmembrane segment of ST specifies Golgi retention. Furthermore, altering this segment in full-length ST results in the protein accumulating on the cell surface. However, the retaining effect of the transmembrane domain of ST is augmented by the presence of adjacent lumenal and cytoplasmic sequences from ST. If these sequences are spaced apart by a transmembrane domain of the same length as that of ST they too can specify Golgi retention. Thus retention in the Golgi of ST appears to involve recognition of an extended region of the protein within and on both sides of the bilayer.


title: Vaccinia virus late transcripts generated in vitro have a poly(A) head.

text: A cell free system mediating accurate transcription of vaccinia virus genes was established using lysates of cells in the late phase of infection. Vaccinia late genes are faithfully transcribed in this extract whereas cellular pol II and pol III promoters are not recognized. The late viral transcripts contain a poly(A) head of approximately 35 nt at the 5' end which is not co-linearly encoded in the externally added template. The transcripts obtained in vitro are indistinguishable from the mature in vivo RNAs. The poly(A) head is synthesized de novo and its formation appears to be directly coupled to the transcription of the gene. The synthesis of the poly(A) head transcripts in vitro is consistent with a proposed slippage model.


title: Recombination between satellite RNAs of turnip crinkle virus.

text: Turnip crinkle virus (TCV) is associated with satellite (sat) RNAs (sat-RNA D, sat-RNA F), defective interfering (DI) RNAs (DI RNA G, DI1 RNA), and one RNA with properties of both sat-RNAs and DI RNAs (sat-RNA C). When plants were inoculated with TCV, sat-RNA D and in vitro sat-RNA C transcripts containing non-viable mutations in the 5' domain, recombinant sat-RNAs were recovered. These recombinants were composed of sat-RNA D at the 5' end and sat-RNA C sequences at the 3' end. Analysis of 20 independent recombination junctions revealed that unequal crossing-over had occurred in planta in a region of sequence similarity between the two sat-RNAs which resulted in the duplication of 3-16 nucleotides. Thirty percent of the sat-RNA recombinants also had one to three additional nucleotides inserted at the crossover junctions which did not correspond to either sat-RNA C or sat-RNA D sequence. The right side of the recombination junctions always began with one of three consecutive nucleotides of sat-RNA C. Based on the similarity between this sequence of sat-RNA C, the right side junction of DI RNA G and the 5' end of TCV, as well as the sequence similarity between right side junctions of DI1 RNA and sat-RNA C and the 5' end of the sat-RNAs, a replicase-driven copy choice mechanism is proposed.(ABSTRACT TRUNCATED AT 250 WORDS)


title: Frame shift mutations as a novel mechanism for the generation of neutralization resistant mutants of human respiratory syncytial virus.

text: The genetic characterization of four previously reported mutants of human respiratory syncytial (RS) virus resistant to monoclonal antibody 63G is described. Sequences of the G protein genes were obtained from: (i) mRNA derived cDNA recombinants, (ii) direct mRNA sequencing and (iii) amplified vRNA derived cDNAs. The results obtained indicate that the original escape mutants, recovered from individual plaques, contained heterogeneous viral populations. This heterogeneity affected the number of adenosine residues present after nucleotides 588 or 623 of the G protein gene. Mutant viruses recovered after a second plaque purification step generated homogeneous sequences but contained single adenosine insertions or deletions at those two sites compared with the Long sequence. These genetic alterations introduced frameshift changes which are reflected in both the antigenic and structural properties of the mutant G proteins. The origin and importance of frameshift mutations in the RS virus G protein gene are discussed.


title: Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide.

text: Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids. To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein. This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells. To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells. The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis. Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons.


title: Messenger RNAs of a strongly-expressed late gene of cowpox virus contain 5'-terminal poly(A) sequences.

text: We have identified and characterized one of the most strongly-expressed genes of cowpox virus (CPV). This is the gene encoding the major protein component of the A-type inclusion bodies produced by this virus. This gene (designated the 160K gene) is transcribed late during the infection. Analyses of its mRNAs showed that these late RNAs, unlike all other characterized late mRNAs of poxviruses, are uniform in length. However, the most remarkable feature of the mRNAs of the 160K gene is the structure of their 5'-termini. Most of these mRNAs have 5'-terminal poly(A) sequences containing 5-21 residues. Furthermore, these 5'-terminal poly(A) sequences are not complementary to the corresponding region of the template strand of the viral DNA. Instead, the nucleotide sequences of the mRNA and the viral DNA diverge at the site of the three As in the sequence 5'-TAAATG-3' containing the gene's initiation codon. Consequently, the poly(A) provides the leader sequences of these mRNAs. These unusual 5'-terminal structures suggest that the late mRNAs of pox-virus genes are generated by a novel process.


title: Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

text: The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein.


title: The carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1.

text: Two size classes of O-glycosidically linked oligosaccharides were liberated from glycoprotein E1 of mouse hepatitis virus (MHV) A59 by reductive beta-elimination and separated by h.p.l.c. The structures of the reduced oligosaccharides were determined by successive exoglycosidase digestions and by methylation analyses involving combined capillary gas chromatography-mass spectrometry and mass fragmentography after chemical ionization with ammonia. Oligosaccharide A (Neu5Ac alpha 2----3 Gal beta 1----3 GalNAc) comprised 35% of the total carbohydrate side chains, while the remaining 65% of the oligosaccharides of E1 had the branched structure B: Neu5Ac alpha 2----3 Gal beta 1----3 (Neu5Ac alpha 2----6) GalNAc. Both oligosaccharides were linked to the E1 polypeptide via N-acetylgalactosamine, and 20% of the sialic acids present in E1 glycopeptides were found to consist of N-acetyl-9-mono-O-acetylneuraminic acid. The reported structures of the O-linked glycans are discussed in the context of the amino acid sequence of E1, which exhibits a cluster of four hydroxyamino acids (Ser-Ser-Thr-Thr) as potential O-glycosylation sites at the amino terminus. Oligosaccharides with identical structures and an identical O-glycosylated tetrapeptide sequence are present in the blood group M-active glycophorin A of the human erythrocyte membrane.


title: Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187.

text: At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.


title: Interactions of phage P22 tails with their cellular receptor, Salmonella O-antigen polysaccharide.

text: Bacteriophage P22 binds to its cell surface receptor, the repetitive O-antigen structure in Salmonella lipopolysaccharide, by its six homotrimeric tailspikes. Receptor binding by soluble tailspikes and the receptor-inactivating endorhamnosidase activity of the tailspike protein were studied using octa- and dodecasaccharides comprising two and three O-antigen repeats of Salmonella enteritidis and Salmonella typhimurium lipopolysaccharides. Wild-type tailspike protein and three mutants (D392N, D395N, and E359Q) with defective endorhamnosidase activity were used. Oligosaccharide binding to all three subunits, measured by a tryptophan fluorescence quench or by fluorescence depolarization of a coumarin label attached to the reducing end of the dodecasaccharide, occurs independently. At 10 degrees C, the binding affinities of all four proteins to oligosaccharides from both bacterial strains are identical within experimental error, and the binding constants for octa- and dodecasaccharides are 1 x 10(6) M(-1) and 2 x 10(6) M(-1), proving that two O-antigen repeats are sufficient for lipopolysaccharide recognition by the tailspike. Equilibration with the oligosaccharides occurs rapidly, but the endorhamnosidase produces only one cleavage every 100 s at 10 degrees C or about 2 min(-1) at the bacterial growth temperature. Thus, movement of virions in the lipopolysaccharide layer before DNA injection may involve the release and rebinding of individual tailspikes rather than hydrolysis of the O-antigen.


The capacity to change: building global environmental health expertise.


Conservation medicine: combining the best of all worlds.


Infectious disease: the human costs of our environmental errors.


title: Role of spleen macrophages in innate and acquired immune responses against mouse hepatitis virus strain A59.

text: Owing to their scavenging and phagocytic functions, spleen macrophages are regarded to be important in the induction and maintenance of both innate and acquired immune defence mechanisms. In this study, we investigated the role of spleen macrophages in immunity against mouse hepatitis virus strain A59 (MHV-A59). Previous studies showed that spleen and liver macrophages are the first target cells for infection by MHV-A59 in vivo, suggesting that they could be involved in the induction of immune responses against MHV-A59. We used a macrophage depletion technique to deplete macrophages in vivo and studied the induction of virus-specific antibody and cytotoxic T-cell (CTL) responses and non-immune resistance against MHV-A59 in normal and macrophage-depleted mice. Virus titres in spleen and liver increased rapidly in macrophage-depleted mice, resulting in death of mice within 4 days after infection. Elimination of macrophages before immunization with MHV-A59 resulted in increased virus-specific humoral and T-cell proliferative responses. However, virus-specific CTL responses were not altered in macrophage-depleted mice. Our results show that spleen macrophages are of major importance as scavenger cells during MHV-A59 infection and are involved in clearance of virus from the host. In addition, macrophages may be involved in the regulation of acquired immune responses. In the absence of macrophages, increased virus-specific T-cell and antibody responses are detectable, suggesting that macrophages suppress MHV-A59-specific T- and B-cell responses and that other cells serve as antigen-presenting cells.


title: Effect of adoptive transfer of CD4, CD8 and B cells on recovery from MHV3-induced immunodeficiencies.

text: A chronic viral infection can occur when the host fails to mount an effective immune response to clear the virus. Mouse hepatitis virus type 3 (MHV3) appears to be an excellent model for the study of the relationship between viral-induced immunodeficiency and chronic disease development. (C57BL/6 x A/J)F1 mice surviving acute hepatitis develop a chronic disease characterized by T- and B-cell immunodeficiencies, viral persistence in various organs including the brain, spleen and thymus, and death within 3 months postinfection (p.i.). We have reported that T- or B-cell deficiencies, observed in MHV3 chronically infected (C57BL/6 x A/J)F1 mice, can be partially or totally thwarted by adoptive transfer of CD4+, CD8+ and/or B cells, at 15 days p.i. in mice surviving the acute phase of the disease. Adoptive transfer of syngeneic CD4+ and/or CD8+ allowed a partial restoration of the T-cell deficiencies, as characterized by thymic atrophy, decrease in splenic T cells, and in all thymocyte subpopulations. B-cell immunodeficiency, as defined by a decrease in splenic B cells, as well as in the bone marrow pre-B- and B-cell compartments, and the occurrence of abnormally larger forms of bone marrow pre-B and B cells, were partially thwarted by B-cell treatment only. Splenic B cells and the bone marrow B-cell compartment, respectively, returned partially or totally to normal values, whereas the pre-B-cell compartment remained depleted in infected mice treated with B cells. Levels of all immunoglobulin classes returned to normal values in MHV3 chronically infected mice when treated with CD4+ in combination with CD8+ cells. All T- and/or B-cell treatments, however, were sufficient to thwart the process of the chronic disease, and favoured the survival of mice for up to 6 months p.i.


23rd Annual Meeting


docid: pdnkabwj

title: The etiology of Balkan endemic nephropathy: still more questions than answers.

text: Balkan endemic nephropathy (BEN) has attracted increasing attention as a possible environmental disease, and a significant amount of research from complementary scientific fields has been dedicated to its etiology. There are two actual competing theories attempting to explain the cause of this kidney disease: 1) the mycotoxin hypothesis, which considers that BEN is produced by ochratoxin A ingested intermittently in small amounts by the individuals in the endemic regions, and 2) the Pliocene lignite hypothesis, which proposes that the disease is caused by long-term exposure to polycyclic aromatic hydrocarbons and other toxic organic compounds leaching into the well drinking water from low rank coals underlying or proximal to the endemic settlements. We outline the current developments and future prospects in the study of BEN and differentiate possible factors and cofactors in disease etiology.


title: Evaluation of a model for post-partum arthritis and the role of oestrogen in prevention of MRL-lpr associated rheumatic conditions.

text: Sixty-eight percent of female MRL-lpr mice developed a post-partum exacerbation of their mild spontaneous arthritis within 30 days of parturition. The flare became evident between 5 and 15 days after delivery. Histologically it was characterized by a significant increase of subsynovial inflammation and synovial hyperplasia without changes in the level of cartilage and bone erosion. Immunohistologically, marked subsynovial and frequent synovial staining of MHC class II bearing cells was noted, along with the sporadic presence of CD3, CD4, and CD43 receptor-bearing cells in the subsynovium. Injection of physiological levels (0.08 mg/kg) of estradiol on days 2, 3, 9, 15 and 20 post-partum delayed and reduced the flare to 23% of the animals. Administration of pharmacological amounts (0.4 mg/kg per day for 2 weeks following Freund's complete adjuvant injection) prevented adjuvant-enhanced arthritis, reducing the incidence from 67% to the baseline 21% level. Deleterious changes in the underlying systemic lupus erythematosus (SLE), as demonstrated by proteinuria and mortality rate increases, were elicited only by the employed pharmacological amounts of estradiol. These results indicate that the MRL-lpr mice might serve as a model for post-partum flare of arthritis in SLE and rheumatoid arthritis by providing an approach to study the complexity of the effects of pregnancy on autoimmune diseases, and to obtain further evidence for the involvement of oestrogen in arthritis.


title: Avridine-induced arthritis in rats; a T cell-dependent chronic disease influenced both by MHC genes and by non-MHC genes.

text: Avridine is a potent synthetic adjuvant that can induce arthritis is most rat strains. The clinical appearance and histopathology of avridine-induced arthritis show great similarity to other arthritis models such as collagen-induced arthritis. In LEW and DA rats the avridine-induced arthritis is severe and long lasting. To investigate a possible genetic influence on the disease we compared LEW, DA and E3 rats, which are of different genetic origins, for their ability to develop arthritis after injection of a low dose of avridine (1.5 mg/rat). The E3 rat was shown to be resistant, whereas all of the DA rats developed arthritis. Recombinant inbred strains derived from DA and E3 parentals varied in susceptibility to avridine. Only strains sharing RT1av1 with DA developed arthritis, indicating a role for the MHC genes. The MHC association was further analysed in a series of Lewis congenic strains using the 1.5 mg avridine dose. All strains developed arthritis. LEW.1C and LEW.1W developed only acute arthritis, whereas LEW.1A, LEW, LEW.1D, LEW.1N and LEW.1F developed chronic arthritis. In particular, the LEW.1F rats developed a chronic severe arthritis of high incidence. The chronic arthritis showed an active, erosive joint inflammation several months after induction. Nude rats are resistant to avridine-induced arthritis, indicating a T cell dependence of the disease which supports the importance of MHC. However, non-MHC genes are also crucial to arthritis development. Recombinants between DA and E3, sharing RT1av1 with DA, showed either a lower incidence or a lower severity of disease than the DA rats. The E3 rat and the recombinants with RT1u were completely resistant, whereas LEW.1W, also RT1u, were highly susceptible.


title: Abnormalities in subset distribution, activation, and differentiation of T cells isolated from large intestine biopsies in HIV infection. The Berlin Diarrhoea/Wasting Syndrome Study Group.

text: Intestinal T cells have a unique state of activation and differentiation which might specifically affect or be affected by HIV infection. Lymphocyte subsets in the peripheral blood are well characterized, but our knowledge about intestinal lymphocytes in HIV infection is incomplete. We therefore analysed lymphocytes isolated from large intestine biopsies of AIDS patients and controls by three-colour cytofluorometry. In the large intestine of HIV-infected patients CD4 T cells were reduced and CD8 T cells were increased compared with controls. Most of the CD8 T cells in the colorectal mucosa of AIDS patients were of the cytotoxic phenotype. Activated and resting CD4 T cells were similarly reduced, the expression of CD25 and HLA-DR of CD8 T cells was unaltered and increased, respectively. In intestinal CD4 T cells the expression of CD29 was decreased, but the expression of CD45RO and HML-1 was normal. CD8 T cells had a decreased expression of all these differentiation markers. Our findings demonstrate substantial alterations in subset distribution, activation, and differentiation of large intestine T cells, which may contribute to the secondary infections and malignancies commonly observed in the gut of AIDS patients.


title: Vaccination and genetic experiments demonstrate that adjuvant-oil-induced arthritis and homologous type II collagen-induced arthritis in the same rat strain are different diseases.

text: The DA rat is highly susceptible to induction of arthritis after immunization with homologous type II collagen (CII) emulsified in Freund's incomplete adjuvant (FIA), resulting in collagen-induced arthritis (CIA). The DA rat also develops arthritis after injection of FIA alone (oil-induced arthritis (OIA)). This finding allows a direct comparison of two different models for rheumatoid arthritis; one induced with a defined auto-immunogen and one with a pure adjuvant. Both CIA and OIA develop approximately 2 weeks after induction but OIA is a self-limited acute disease whereas CIA induced with homologous CII follows a chronic disease course. Immunization with CII leads to a strong autoantibody response to CII while injection of FIA leads to no or very limited anti-CII antibody response. The Lewis rat develops neither CIA nor OIA while F1 (DA x Lewis) rats develop CIA but not OIA. Olive oil or CII emulsified in olive oil does not induce arthritis in DA rats. Pretreatment with CII in olive oil vaccinates against CIA but not OIA whereas pretreatment with FIA vaccinates against OIA but not CIA. These findings demonstrate that inclusion of CII in the adjuvant leads to a disease distinct from OIA which is characterized by a CII autoimmune response and chronicity of the disease course.


title: T cell memory specific for self and non-self antigens in rats persistently infected with Borna disease virus.

text: We have studied CD4+ Th1 T cell responses in Borna disease (BD), a virus-mediated immune disease of the central nervous system (CNS), and demonstrate the priming of virus-specific as well as autoreactive T cells specific for myelin antigens in the course of viral infection. The fate of these in vivo generated T cells was subsequently assessed by in vitro proliferation assays with lymphocytes from different lymphoid organs of diseased animals over a long period of time. Virus-specific T cell responses continuously decreased during the establishment of persistent infection and could no longer be detected after 5-6 months post infectionem, when inflammatory reactions in the brain had ceased. By contrast, autoantigen-specific T cells kept their ability to mount characteristic secondary responses--although at an overall rather low level--over long periods of time; these autoreactive T cells homed to a specific lymphoid organ, the perithymic lymph node. Our study thus describes for the first time a complete decline of virus-specific T cell memory in a persistent viral infection, and raises the question how long-lasting T cell autoreactivity is controlled.


Enhancement of the Pathogenicity of Mouse Hepatitis Virus (MHV1) By Prior Infection of Mice with Certain Leukaemia Agents


title: AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

text: Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.


title: Biosynthesis and processing of ribophorins in the endoplasmic reticulum

text: Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the unglycosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translationally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demonstrated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosaccharide side chain(s). Direct analysis of the oligosaccharides of ribophorin l showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from Man8GlcNAc to Man5GlcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus.


title: Specific asparagine-linked oligosaccharides are not required for certain neuron-neuron and neuron-Schwann cell interactions

text: To determine whether specific asparagine-linked (N-linked) oligosaccharides present in cell surface glycoproteins are required for cell-cell interactions within the peripheral nervous system, we have used castanospermine to inhibit maturation of N-linked sugars in cell cultures of neurons or neurons plus Schwann cells. Maximally 10-15% of the N-linked oligosaccharides on neuronal proteins have normal structure when cells are cultured in the presence of 250 micrograms/ml castanospermine; the remaining oligosaccharides are present as immature carbohydrate chains not normally found in these glycoproteins. Although cultures were treated for 2 wk with castanospermine, cells always remained viable and appeared healthy. We have analyzed several biological responses of embryonic dorsal root ganglion neurons, with or without added purified populations of Schwann cells, in the presence of castanospermine. We have observed that a normal complement of mature, N- linked sugars are not required for neurite outgrowth, neuron-Schwann cell adhesion, neuron-induced Schwann cell proliferation, or ensheathment of neurites by Schwann cells. Treatment of neuronal cultures with castanospermine increases the propensity of neurites to fasciculate. Extracellular matrix deposition by Schwann cells and myelination of neurons by Schwann cells are greatly diminished in the presence of castanospermine as assayed by electron microscopy and immunocytochemistry, suggesting that specific N-linked oligosaccharides are required for the expression of these cellular functions.


title: Biochemical studies on cell fusion. II. Control of fusion response by lipid alteration

text: The preceding communication (Roos, D.S. and P.W. Choppin, 1985, J. Cell Biol. 101:1578-1590) described the lipid composition of a series of mouse fibroblast cell lines which vary in susceptibility to the fusogenic effects of polyethylene glycol (PEG). Two alterations in lipid content were found to be directly correlated with resistance to PEG-induced cell fusion: increases in fatty acyl chain saturation, and the elevation of neutral glycerides, including an unusual ether-linked compound. In this study, we have probed the association between lipid composition and cell fusion through the use of fatty acid supplements to the cellular growth medium, and show that the fusibility of cells can be controlled by altering their acyl chain composition. The parental Clone 1D cells contain moderately unsaturated fatty acids with a ratio of saturates to polyunsaturates (S/P) approximately 1 and fuse virtually to completion following a standard PEG treatment. By contrast, the lipids of a highly fusion-resistant mutant cell line, F40, are highly saturated (S/P approximately 4). When the S/P ratio of Clone 1D cells was increased to approximate that normally found in F40 cells by growth in the presence of high concentrations of saturated fatty acids, they became highly resistant to PEG. Reduction of the S/P ratio of F40 cells by growth in cis-polyunsaturated fatty acids rendered them susceptible to fusion. Cell lines F8, F16, etc., which are normally intermediate between Clone 1D and F40 in both lipid composition and fusion response, can be altered in either direction (towards either increased or decreased susceptibility to fusion) by the addition of appropriate fatty acids to the growth medium. Although trans-unsaturated fatty acids have phase-transition temperatures roughly similar to saturated compounds, and might therefore be expected to affect membrane fluidity in a similar manner, trans-unsaturated fatty acids exerted the same effect as cis-unsaturates on the control of PEG-induced cell fusion. This observation suggests that the control of cell fusion by alteration of fatty acid content is not due to changes in membrane fluidity, and thus that the fatty acids are involved in some other way in the modulation of cell fusion.