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Science is humanity's best insurance against threats from nature, but it is a fragile enterprise that must be nourished and protected. The preponderance of scientific evidence indicates a natural origin for SARS-CoV-2. Yet, the theory that SARS-CoV-2 was engineered in and escaped from a lab dominates media attention, even in the absence of strong evidence. We discuss how the resulting anti-science movement puts the research community, scientific research, and pandemic preparedness at risk.
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In the United States (US), biosafety and biosecurity oversight of research on viruses is being reappraised. Safety in virology research is paramount and oversight frameworks should be reviewed periodically. Changes should be made with care, however, to avoid impeding science that is essential for rapidly reducing and responding to pandemic threats as well as addressing more common challenges caused by infectious diseases. Decades of research uniquely positioned the US to be able to respond to the COVID-19 crisis with astounding speed, delivering life-saving vaccines within a year of identifying the virus. We should embolden and empower this strength, which is a vital part of protecting the health, economy, and security of US citizens. Herein, we offer our perspectives on priorities for revised rules governing virology research in the US.
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Pesquisa Biomédica , Contenção de Riscos Biológicos , Virologia , Humanos , COVID-19 , Estados Unidos , Vírus , Pesquisa Biomédica/normasRESUMO
Viruses have brought humanity many challenges: respiratory infection, cancer, neurological impairment and immunosuppression to name a few. Virology research over the last 60+ years has responded to reduce this disease burden with vaccines and antivirals. Despite this long history, the COVID-19 pandemic has brought unprecedented attention to the field of virology. Some of this attention is focused on concern about the safe conduct of research with human pathogens. A small but vocal group of individuals has seized upon these concerns - conflating legitimate questions about safely conducting virus-related research with uncertainties over the origins of SARS-CoV-2. The result has fueled public confusion and, in many instances, ill-informed condemnation of virology. With this article, we seek to promote a return to rational discourse. We explain the use of gain-of-function approaches in science, discuss the possible origins of SARS-CoV-2 and outline current regulatory structures that provide oversight for virological research in the United States. By offering our expertise, we - a broad group of working virologists - seek to aid policy makers in navigating these controversial issues. Balanced, evidence-based discourse is essential to addressing public concern while maintaining and expanding much-needed research in virology.
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Pesquisa , Virologia , Viroses , Humanos , COVID-19/prevenção & controle , Disseminação de Informação , Pandemias/prevenção & controle , Formulação de Políticas , Pesquisa/normas , Pesquisa/tendências , SARS-CoV-2 , Virologia/normas , Virologia/tendências , Viroses/prevenção & controle , Viroses/virologia , VírusRESUMO
BACKGROUND: Emergency department (ED) clinicians are more frequently providing care, including end-of-life care, to older people. OBJECTIVES: To estimate the need for ED end-of-life care for people aged ≥65 years, describe characteristics of those dying within 48 hours of ED presentation and compare those dying in ED with those dying elsewhere. METHODS: We conducted a retrospective cohort study analysing data from 177 hospitals in Australia and New Zealand. Data on older people presenting to ED from January to December 2018, and those who died within 48 hours of ED presentation, were analysed using simple descriptive statistics and univariate logistic regression. RESULTS: From participating hospitals in Australia or New Zealand, 10,921 deaths in older people occurred. The 48-hour mortality rate was 6.43 per 1,000 ED presentations (95% confidence interval: 6.31-6.56). Just over a quarter (n = 3,067, 28.1%) died in ED. About one-quarter of the cohort (n = 2,887, 26.4%) was triaged into less urgent triage categories. Factors with an increased risk of dying in ED included age 65-74 years, ambulance arrival, most urgent triage categories, principal diagnosis of circulatory system disorder, and not identifying as an Aboriginal or Torres Strait Islander person. Of the 7,677 older people admitted, half (n = 3,836, 50.0%) had an encounter for palliative care prior to, or during, this presentation. CONCLUSIONS: Our findings provide insight into the challenges of recognising the dying older patient and differentiating those appropriate for end-of-life care. We support recommendations for national advanced care planning registers and suggest a review of triage systems with an older person-focused lens.
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Serviço Hospitalar de Emergência , Hospitalização , Idoso , Humanos , Austrália/epidemiologia , Nova Zelândia/epidemiologia , Estudos RetrospectivosRESUMO
BACKGROUND: Patients receiving palliative care are often on complex medication regimes to manage their symptoms and comorbidities and at high risk of medication-related problems. The aim of this cross-sectional study was to evaluate the involvement of a pharmacist to an existing community specialist palliative care telehealth service on patients' medication management. METHOD: The specialist palliative care pharmacist attended two palliative care telehealth sessions per week over a six-month period (October 2020 to March 2021). Attendance was allocated based on funding received. Data collected from the medication management reviews included prevalence of polypharmacy, number of inappropriate medication according to the Screening Tool of Older Persons Prescriptions in Frail adults with limited life expectancy criteria (STOPP/FRAIL) and recommendations on deprescribing, symptom control and medication management. RESULTS: In total 95 patients participated in the pharmaceutical telehealth service with a mean age of 75.2 years (SD 10.67). Whilst 81 (85.3%) patients had a cancer diagnosis, 14 (14.7%) had a non-cancer diagnosis. At referral, 84 (88.4%, SD 4.57) patients were taking ≥ 5 medications with 51 (53.7%, SD 5.03) taking ≥ 10 medications. According to STOPP/FRAIL criteria, 142 potentially inappropriate medications were taken by 54 (56.8%) patients, with a mean of 2.6 (SD 1.16) inappropriate medications per person. Overall, 142 recommendations were accepted from the pharmaceutical medication management review including 49 (34.5%) related to deprescribing, 20 (14.0%) to medication-related problems, 35 (24.7%) to symptom management and 38 (26.8%) to medication administration. CONCLUSION: This study provided evidence regarding the value of including a pharmacist in palliative care telehealth services. Input from the pharmacist resulted in improved symptom management of community palliative care patients and their overall medication management.
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Cuidados Paliativos , Farmacêuticos , Telemedicina , Humanos , Estudos Transversais , Cuidados Paliativos/métodos , Cuidados Paliativos/normas , Idoso , Telemedicina/normas , Feminino , Masculino , Idoso de 80 Anos ou mais , Conduta do Tratamento Medicamentoso/normas , Polimedicação , Pessoa de Meia-IdadeRESUMO
Viruses emerging from wildlife can cause outbreaks in humans and domesticated animals. Predicting the emergence of future pathogens and mitigating their impacts requires an understanding of what shapes virus diversity and dynamics in wildlife reservoirs. In order to better understand coronavirus ecology in wild species, we sampled birds within a coastal freshwater lagoon habitat across 5 years, focussing on a large population of mute swans (Cygnus olor) and the diverse species that they interact with. We discovered and characterised the full genome of a divergent gammacoronavirus belonging to the Goose coronavirus CB17 species. We investigated the genetic diversity and dynamics of this gammacoronavirus using untargeted metagenomic sequencing of 223 faecal samples from swans of known age and sex, and RT-PCR screening of 1632 additional bird samples. The virus circulated persistently within the bird community; virus prevalence in mute swans exhibited seasonal variations, but did not change with swan age-class or epidemiological year. One whole genome was fully characterised, and revealed that the virus originated from a recombination event involving an undescribed gammacoronavirus species. Multiple lineages of this gammacoronavirus co-circulated within our study population. Viruses from this species have recently been detected in aquatic birds from both the Anatidae and Rallidae families, implying that host species habitat sharing may be important in shaping virus host range. As the host range of the Goose coronavirus CB17 species is not limited to geese, we propose that this species name should be updated to 'Waterbird gammacoronavirus 1'. Non-invasive sampling of bird coronaviruses may provide a tractable model system for understanding the evolutionary and cross-species dynamics of coronaviruses.
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Anseriformes , Infecções por Coronavirus , Coronavirus , Gammacoronavirus , Humanos , Animais , Gammacoronavirus/genética , Coronavirus/genética , Surtos de Doenças , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/veterinária , Animais Selvagens , Variação Genética , Recombinação GenéticaRESUMO
Eight infectious bursal disease virus (IBDV) genogroups have been identified based on the sequence of the capsid hypervariable region (HVR) (A1 to A8). Given reported vaccine failures, there is a need to evaluate the ability of vaccines to neutralize the different genogroups. To address this, we used a reverse genetics system and the chicken B-cell line DT40 to rescue a panel of chimeric IBDVs and perform neutralization assays. Chimeric viruses had the backbone of a lab-adapted strain (PBG98) and the HVRs from diverse field strains as follows: classical F52-70 (A1), U.S. variant Del-E (A2), Chinese variant SHG19 (A2), very virulent UK661 (A3), M04/09 distinct (A4), Italian ITA-04 (A6), and Australian variant Vic-01/94 (A8). Rescued viruses showed no substitutions at amino acid positions 253, 284, or 330, previously found to be associated with cell-culture adaptation. Sera from chickens inoculated with wild-type (wt) (F52-70) or vaccine (228E) A1 strains had the highest mean virus neutralization (VN) titers against the A1 virus (log2 15.4 and 12.7) and the lowest against A2 viruses (log2 7.4 to 7.9; P = 0.0001 to 0.0274), consistent with A1 viruses being most antigenically distant from A2 strains, which correlated with the extent of differences in the predicted HVR structure. VN titers against the other genogroups ranged from log2 9.3 to 13.3, and A1 strains were likely more closely antigenically related to genogroups A3 and A4 than A6 and A8. Our data are consistent with field observations and validate the new method, which can be used to screen future vaccine candidates for breadth of neutralizing antibodies and evaluate the antigenic relatedness of different genogroups. IMPORTANCE There is a need to evaluate the ability of vaccines to neutralize diverse IBDV genogroups and to better understand the relationship between HVR sequence, structure, and antigenicity. Here, we used a chicken B-cell line to rescue a panel of chimeric IBDVs with the HVR from seven diverse IBDV field strains and to conduct neutralization assays and protein modeling. We evaluated the ability of sera from vaccinated or infected birds to neutralize the different genogroups. Our novel chicken B-cell rescue system and neutralization assay can be used to screen IBDV vaccine candidates, platforms, and regimens for the breadth of neutralizing antibody responses elicited, evaluate the antigenic relatedness of diverse IBDV strains, and when coupled with structural modeling, elucidate immunodominant and conserved epitopes to strategically design novel IBDV vaccines in the future.
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Anticorpos Neutralizantes , Infecções por Birnaviridae , Vírus da Doença Infecciosa da Bursa , Doenças das Aves Domésticas , Animais , Austrália , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/virologia , Galinhas , Epitopos , Genótipo , Doenças das Aves Domésticas/imunologiaRESUMO
To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.
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Infecções por Birnaviridae , Birnaviridae , Vírus da Doença Infecciosa da Bursa , Doenças das Aves Domésticas , Compartimentos de Replicação Viral , Replicação Viral , Animais , Birnaviridae/fisiologia , Linhagem Celular , Galinhas/genética , Vírus da Doença Infecciosa da Bursa/fisiologia , Microscopia Eletrônica , RNA Viral/genética , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismoRESUMO
The use of infectious bursal disease virus (IBDV) reverse genetics to engineer tagged reporter viruses has revealed that the virus factories (VFs) of the Birnaviridae family are biomolecular condensates that show properties consistent with liquid-liquid phase separation (LLPS). Although the VFs are not bound by membranes, it is currently thought that viral protein 3 (VP3) initially nucleates the formation of the VF on the cytoplasmic leaflet of early endosomal membranes, and likely drives LLPS. In addition to VP3, IBDV VFs contain VP1 (the viral polymerase) and the dsRNA genome, and they are the sites of de novo viral RNA synthesis. Cellular proteins are also recruited to the VFs, which are likely to provide an optimal environment for viral replication; the VFs grow due to the synthesis of the viral components, the recruitment of other proteins, and the coalescence of multiple VFs in the cytoplasm. Here, we review what is currently known about the formation, properties, composition, and processes of these structures. Many open questions remain regarding the biophysical nature of the VFs, as well as the roles they play in replication, translation, virion assembly, viral genome partitioning, and in modulating cellular processes.
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Birnaviridae , Vírus da Doença Infecciosa da Bursa , Birnaviridae/metabolismo , Compartimentos de Replicação Viral , Linhagem Celular , Replicação Viral , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas Estruturais Virais/metabolismoRESUMO
The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 µm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 µm/s at 16 hpi compared to 0.22 µm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence.IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae This study provides new information that adds to our understanding of dsRNA virus VF trafficking.
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Vírus da Doença Infecciosa da Bursa/genética , Vírus Reordenados/genética , Replicação Viral/genética , Animais , Linhagem Celular , Coinfecção/metabolismo , Citoplasma , Vírus de RNA/genética , Vírus Reordenados/metabolismo , Proteínas Estruturais Virais/genéticaRESUMO
BACKGROUND: The highest healthcare expenditures occur towards the end of life. Costs relate to hospital admissions and investigations to diagnose, prognosticate and direct treatment. AIMS: AnAustralian study to compare the cost of investigations in the last 72 h of life between an inpatient palliative care unit (PCU) and a tertiary hospital. METHOD: We retrospectively reviewed 50 adult medical and surgical patients (admitted for >72 h and who died in hospital) from the PCU and referring tertiary centre between March and July 2016. Patients in the emergency department, intensive care, medical assessment and paediatric and obstetric units were excluded. All patients had an acute resuscitation plan and were on the 'Care of the Dying' pathway. RESULTS: Expenditure was less if palliative care were the primary caregivers, with statistically significant differences in the amount of imaging (P < 0.001) and pathology (P < 0.001) ordered. There was no difference in microbiology (P = 0.172) and histology (P ~ 1) ordered. Total cost of investigations for PCU patients was $1340.60 (4 of 50 patients) compared with $9467.78 (29 of 50 patients) in the tertiary hospital. PCU patients had longer lengths of stay (15.54 days vs 11.06 days) but cost less per bed day ($868.32 vs $878.79 respectively). CONCLUSION: Inpatient PCU are less likely to order investigations and are more cost-effective. A prospective study comparing an inpatient PCU and patients at a tertiary centre, with and without consult liaison palliative care input, would be worthwhile to see if outcomes remain the same and if consult liaison palliative care affects the investigative burden.
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Gastos em Saúde , Cuidados Paliativos , Adulto , Criança , Morte , Hospitais , Humanos , Pacientes Internados , Estudos Prospectivos , Estudos RetrospectivosRESUMO
The Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic betacoronavirus that was first detected in humans in 2012 as a cause of severe acute respiratory disease. As of July 28, 2017, there have been 2,040 confirmed cases with 712 reported deaths. While many infections have been fatal, there have also been a large number of mild or asymptomatic cases discovered through monitoring and contact tracing. New Zealand white rabbits are a possible model for asymptomatic infection with MERS-CoV. In order to discover more about non-lethal infections and to learn whether a single infection with MERS-CoV would protect against reinfection, we inoculated rabbits with MERS-CoV and monitored the antibody and inflammatory response. Following intranasal infection, rabbits developed a transient dose-dependent pulmonary infection with moderately high levels of viral RNA, viral antigen, and perivascular inflammation in multiple lung lobes that was not associated with clinical signs. The rabbits developed antibodies against viral proteins that lacked neutralizing activity and the animals were not protected from reinfection. In fact, reinfection resulted in enhanced pulmonary inflammation, without an associated increase in viral RNA titers. Interestingly, passive transfer of serum from previously infected rabbits to naïve rabbits was associated with enhanced inflammation upon infection. We further found this inflammation was accompanied by increased recruitment of complement proteins compared to primary infection. However, reinfection elicited neutralizing antibodies that protected rabbits from subsequent viral challenge. Our data from the rabbit model suggests that people exposed to MERS-CoV who fail to develop a neutralizing antibody response, or persons whose neutralizing antibody titers have waned, may be at risk for severe lung disease on re-exposure to MERS-CoV.
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Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Coronavirus/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Imuno-Histoquímica , Inflamação/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Testes de Neutralização , Reação em Cadeia da Polimerase , CoelhosRESUMO
[This corrects the article DOI: 10.1371/journal.ppat.1003971.].
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Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.
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Bolsa de Fabricius/citologia , Galinhas , Vírus da Doença Infecciosa da Bursa/fisiologia , Cultura de Vírus/veterinária , Animais , Sobrevivência Celular , Células Cultivadas , Acetato de Tetradecanoilforbol/farmacologia , Cultura de Vírus/métodosRESUMO
Infectious bursal disease virus (IBDV) belongs to the family Birnaviridae and is economically important to the poultry industry worldwide. IBDV infects B cells in the bursa of Fabricius (BF), causing immunosuppression and morbidity in young chickens. In addition to strains that cause classical Gumboro disease, the so-called 'very virulent' (vv) strain, also in circulation, causes more severe disease and increased mortality. IBDV has traditionally been controlled through the use of live attenuated vaccines, with attenuation resulting from serial passage in non-lymphoid cells. However, the factors that contribute to the vv or attenuated phenotypes are poorly understood. In order to address this, we aimed to investigate host cell-IBDV interactions using a recently described chicken primary B-cell model, where chicken B cells are harvested from the BF and cultured ex vivo in the presence of chicken CD40L. We demonstrated that these cells could support the replication of IBDV when infected ex vivo in the laboratory. Furthermore, we evaluated the gene expression profiles of B cells infected with an attenuated strain (D78) and a very virulent strain (UK661) by microarray. We found that key genes involved in B-cell activation and signalling (TNFSF13B, CD72 and GRAP) were down-regulated following infection relative to mock, which we speculate could contribute to IBDV-mediated immunosuppression. Moreover, cells responded to infection by expressing antiviral type I IFNs and IFN-stimulated genes, but the induction was far less pronounced upon infection with UK661, which we speculate could contribute to its virulence.
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Linfócitos B/virologia , Infecções por Birnaviridae/virologia , Galinhas/virologia , Expressão Gênica/genética , Vírus da Doença Infecciosa da Bursa/genética , Doenças das Aves Domésticas/virologia , Virulência/genética , Animais , Bolsa de Fabricius/virologia , Vacinas Atenuadas/imunologiaRESUMO
The National Synchrotron Light Source (NSLS) ceased operation in September 2014 and was succeeded by NSLS-II. There were four in-vacuum undulators (IVUs) in operation at NSLS. The most recently constructed IVU for NSLS was the mini-gap undulator (MGU-X25, to be renamed IVU18 for NSLS-II), which was constructed in 2006. This device was selected to be reused for the New York Structural Biology Consortium Microdiffraction beamline at NSLS-II. At the time of construction, IVU18 was a state-of-the-art undulator designed to be operated as a cryogenic permanent-magnet undulator. Due to the more stringent field quality and impedance requirements of the NSLS-II ring, the transition region was redesigned. The control system was also updated to NSLS-II specifications. This paper reports the details of the IVU18 refurbishment activities including additional magnetic measurement and tuning.
RESUMO
The incorporation of unnatural amino acids (uAA) can introduce novel functional groups into proteins site-specifically, with important applications in basic sciences and protein engineering. However, uAA incorporation can impact protein expression and functional activity depending on its location within the protein-a process that is not yet completely understood and difficult to predict. Therefore, practical applications often necessitate a time-consuming optimization of uAA location by individual gene cloning, expressions, purification, and evaluations for each location tested. To address this limitation, we introduce a streamlined and versatile in vitro system to rapidly express and screen uAA-containing proteins without cumbersome cell culturing or purification procedures. We utilized this technology to simultaneously screen 24 different t4-lysozyme mutants with different uAA incorporation sites in a matter of hours, compared to weeks-long workflow of conventional methods. Screening data offered a mechanistic explanation to some effects of uAA incorporation on expression and activity. Despite these insights, rational prediction of such effects remained challenging, further confirming the value of a rapid screening approach. Biotechnol. Bioeng. 2017;114: 2412-2417. © 2017 Wiley Periodicals, Inc.
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Aminoácidos/genética , Aminoácidos/metabolismo , Perfilação da Expressão Gênica/métodos , Ensaios de Triagem em Larga Escala/métodos , Biossíntese de Proteínas/fisiologia , Frações Subcelulares/metabolismo , Engenharia de Proteínas/métodosRESUMO
Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.