Triple para-substitution reactions of B(C6F5)3 and [(C6F5)3PF]+ with P(SiMe3)3.

para-Substitution reactions on C6F5 rings of Lewis acids have been exploited to achieve triply substituted derivatives. The reaction of B(C6F5)3 with P(SiMe3)3 ultimately affords the Lewis acid B(C6F4P(SiMe3)2)31. This species binds Lewis bases affording the adducts LB(C6F4P(SiMe3)2)3 (L = MeCN 2, OPEt33, PMe34, PBu35) and reacts with LiMe to give the salt [Li][MeB(C6F4P(SiMe3)2)3]·3THF 6. It also reacts with H2O to give (L)B(C6F4PH2)3 (L = H2O 7, MeCN 8). In an analogous fashion, [(C6F5)3PF][B(C6F5)4] was converted to [FP(C6F4P(SiMe3)2)3] [B(C6F5)4] 9 and subsequently to [(MeO)P(C6F4PH2)3][B(C6F5)4] 10.

Phylogenomic analyses and host range prediction of cluster P mycobacteriophages.

Bacteriophages, infecting bacterial hosts in every environment on our planet, are a driver of adaptive evolution in bacterial communities. At the same time, the host range of many bacteriophages-and thus one of the selective pressures acting on complex microbial systems in nature-remains poorly characterized. Here, we computationally inferred the putative host ranges of 40 cluster P mycobacteriophages, including members from 6 subclusters (P1-P6). A series of comparative genomic analyses revealed that mycobacteriophages of subcluster P1 are restricted to the Mycobacterium genus, whereas mycobacteriophages of subclusters P2-P6 are likely also able to infect other genera, several of which are commonly associated with human disease. Further genomic analysis highlighted that the majority of cluster P mycobacteriophages harbor a conserved integration-dependent immunity system, hypothesized to be the ancestral state of a genetic switch that controls the shift between lytic and lysogenic life cycles-a temperate characteristic that impedes their usage in antibacterial applications.

Complete Genome Sequence of the Cluster P Mycobacteriophage Phegasus.

We characterized the complete genome of the cluster P mycobacteriophage Phegasus. Its 47.5-kb genome contains 81 protein-coding genes, 36 of which could be assigned a putative function. Phegasus is most closely related to two subcluster P1 bacteriophages, Mangethe and Majeke, with an average nucleotide identity of 99.63% each.

Resistance properties and the role of the inner membrane and coat of Bacillus subtilis spores with extreme wet heat resistance.

AIMS: A protein termed 2Duf greatly increases wet heat resistance of Bacillus subtilis spores. The current work examines the effects of 2Duf on spore resistance to other sporicides, including chemicals that act on or must cross spores' inner membrane (IM), where 2Duf is likely present. The overall aim was to gain a deeper understanding of how 2Duf affects spore resistance, and of spore resistance itself. METHODS AND RESULTS: 2Duf's presence increased spore resistance to chemicals that damage or must cross the IM to kill spores. Spore coat removal decreased 2Duf-spore resistance to chemicals and wet heat, and 2Duf-spores made at higher temperatures were more resistant to wet heat and chemicals. 2Duf-less spores lacking coats and Ca-dipicolinic acid were also extremely sensitive to wet heat and chemicals that transit the IM to kill spores. CONCLUSIONS: The new work plus previous results lead to a number of important conclusions as follows. (1) 2Duf may influence spore resistance by decreasing the permeability of and lipid mobility in spores' IM. (2) Since wet heat-killed spores that germinate do not accumulate ATP, wet heat may inactivate some spore IM protein essential in ATP production which is stabilized in a more rigid IM. (3) Both Ca-dipicolinic acid and the spore coat play an important role in the permeability of the spore IM, and thus in many spore resistance properties. SIGNIFICANCE AND IMPACT OF THE STUDY: The work in this manuscript gives a new insight into mechanisms of spore resistance to chemicals and wet heat, to the understanding of spore wet heat killing, and the role of Ca-dipicolinic acid and the coat in spore resistance.

Electronic data collection, management and analysis tools used for outbreak response in low- and middle-income countries: a systematic review and stakeholder survey.

BACKGROUND: Use of electronic data collection, management and analysis tools to support outbreak response is limited, especially in low income countries. This can hamper timely decision-making during outbreak response. Identifying available tools and assessing their functions in the context of outbreak response would support appropriate selection and use, and likely more timely data-driven decision-making during outbreaks. METHODS: We conducted a systematic review and a stakeholder survey of the Global Outbreak Alert and Response Network and other partners to identify and describe the use of, and technical characteristics of, electronic data tools used for outbreak response in low- and middle-income countries. Databases included were MEDLINE, EMBASE, Global Health, Web of Science and CINAHL with publications related to tools for outbreak response included from January 2010-May 2020. Software tool websites of identified tools were also reviewed. Inclusion and exclusion criteria were applied and counts, and proportions of data obtained from the review or stakeholder survey were calculated. RESULTS: We identified 75 electronic tools including for data collection (33/75), management (13/75) and analysis (49/75) based on data from the review and survey. Twenty-eight tools integrated all three functionalities upon collection of additional information from the tool developer websites. The majority were open source, capable of offline data collection and data visualisation. EpiInfo, KoBoCollect and Open Data Kit had the broadest use, including for health promotion, infection prevention and control, and surveillance data capture. Survey participants highlighted harmonisation of data tools as a key challenge in outbreaks and the need for preparedness through training front-line responders on data tools. In partnership with the Global Health Network, we created an online interactive decision-making tool using data derived from the survey and review. CONCLUSIONS: Many electronic tools are available for data -collection, -management and -analysis in outbreak response, but appropriate tool selection depends on knowledge of tools' functionalities and capabilities. The online decision-making tool created to assist selection of the most appropriate tool(s) for outbreak response helps by matching requirements with functionality. Applying the tool together with harmonisation of data formats, and training of front-line responders outside of epidemic periods can support more timely data-driven decision making in outbreaks.

The geographic distribution of un-immunized children in Ontario, Canada: Hotspot detection using Bayesian spatial analysis.

BACKGROUND: In Ontario, Canada, little is currently known about the extent to which un-immunized children may cluster geographically. Our objectives were to: describe the geographic distribution of fully un-immunized children; identify geographic clusters (hotspots) of un-immunized children; and to characterize the contribution of spatial effects and covariates on hotspots, where found. METHODS: Our analytic cohort consisted of Ontario students aged 7-17 years in the 2016-2017 school year. We defined students as un-immunized if they had zero doses of any vaccine and a non-medical exemption recorded in Ontario's registry. We calculated unadjusted proportions of un-immunized students by Census Subdivision (CSD) and then used a sequential approach to identify hotspots starting first with hotspot identification at the CSD level and then probed identified hotspots further by Dissemination Area (DA) and including covariates. Hotspots were identified using the Besag-York-Mollie Bayesian spatial model and were defined as areas with >95% probability of having two times the proportion of un-immunized students, relative to the province overall. RESULTS: We identified 15,208 (0.94%) un-immunized children within our cohort consisting of more than 1.61 million students. Unadjusted proportions of un-immunized students varied greatly by geography, ranging from 0% to 21.5% by CSD. We identified 16 hotspot CSDs which clustered in five distinct areas, all of which were located in southern Ontario. The contribution of covariates and spatial effects on the risk of having un-immunized students varied greatly across hotspot areas. CONCLUSIONS: Although the provincial proportion (0.94%) of un-immunized students is small, geographical clustering of such students is evident in Ontario and in some areas presents an important risk for future outbreaks. Further qualitative work within these hotspot areas would be a helpful next step to better characterize the factors associated with vaccine refusal in these communities.

Treating HIV-associated cytomegalovirus retinitis with oral valganciclovir and intra-ocular ganciclovir by primary HIV clinicians in southern Myanmar: a retrospective analysis of routinely collected data.

BACKGROUND: Cytomegalovirus retinitis (CMVR) is an opportunistic infection in HIV-infected people. Intraocular or intravenous ganciclovir was gold standard for treatment; however, oral valganciclovir replaced this in high-income countries. Low- and middle-income countries (LMIC) frequently use intraocular injection of ganciclovir (IOG) alone because of cost. METHODS: Retrospective review of all HIV-positive patients with CMVR from February 2013 to April 2017 at a Médecins Sans Frontièrs HIV clinic in Myanmar. Treatment was classified as local (IOG) or systemic (valganciclovir, or valganciclovir and IOG). The primary outcome was change in visual acuity (VA) post-treatment. Mortality was a secondary outcome. RESULTS: Fifty-three patients were included. Baseline VA was available for 103 (97%) patient eyes. Active CMVR was present in 72 (68%) eyes. Post-treatment, seven (13%) patients had improvement in VA, 30 (57%) had no change, and three (6%) deteriorated. Among patients receiving systemic therapy, four (12.5%) died, compared with five (24%) receiving local therapy (p = 0.19). CONCLUSIONS: Our results from the first introduction of valganciclovir for CMVR in LMIC show encouraging effectiveness and safety in patients with advanced HIV. We urge HIV programmes to include valganciclovir as an essential medicine, and to include CMVR screening and treatment in the package of advanced HIV care.

The practice of evaluating epidemic response in humanitarian and low-income settings: a systematic review.

BACKGROUND: Epidemics of infectious disease occur frequently in low-income and humanitarian settings and pose a serious threat to populations. However, relatively little is known about responses to these epidemics. Robust evaluations can generate evidence on response efforts and inform future improvements. This systematic review aimed to (i) identify epidemics reported in low-income and crisis settings, (ii) determine the frequency with which evaluations of responses to these epidemics were conducted, (iii) describe the main typologies of evaluations undertaken and (iv) identify key gaps and strengths of recent evaluation practice. METHODS: Reported epidemics were extracted from the following sources: World Health Organization Disease Outbreak News (WHO DON), UNICEF Cholera platform, Reliefweb, PROMED and Global Incidence Map. A systematic review for evaluation reports was conducted using the MEDLINE, EMBASE, Global Health, Web of Science, WPRIM, Reliefweb, PDQ Evidence and CINAHL Plus databases, complemented by grey literature searches using Google and Google Scholar. Evaluation records were quality-scored and linked to epidemics based on time and place. The time period for the review was 2010-2019. RESULTS: A total of 429 epidemics were identified, primarily in sub-Saharan Africa, the Middle East and Central Asia. A total of 15,424 potential evaluations records were screened, 699 assessed for eligibility and 132 included for narrative synthesis. Only one tenth of epidemics had a corresponding response evaluation. Overall, there was wide variability in the quality, content as well as in the disease coverage of evaluation reports. CONCLUSION: The current state of evaluations of responses to these epidemics reveals large gaps in coverage and quality and bears important implications for health equity and accountability to affected populations. The limited availability of epidemic response evaluations prevents improvements to future public health response. The diversity of emphasis and methods of available evaluations limits comparison across responses and time. In order to improve future response and save lives, there is a pressing need to develop a standardized and practical approach as well as governance arrangements to ensure the systematic conduct of epidemic response evaluations in low-income and crisis settings.

High levels of viral repression, malnutrition and second-line ART use in adolescents living with HIV: a mixed methods study from Myanmar.

BACKGROUND: Adolescents living with HIV/AIDS (ALHIV) are a particularly vulnerable but often overlooked group in the HIV response despite additional disease management challenges. METHODS: All ALHIV (10-19 years), on ART for ≥6 months, presenting to care at a Médecins Sans Frontières (MSF) clinic in Myanmar from January-April 2016 were eligible for the quantitative study component (clinical history, medical examination, laboratory investigation). A subset of these respondents were invited to participate in qualitative interviews. Interviews and focus groups were also conducted with other key informants (care givers, clinicians). RESULTS: Of 177 ALHIV, 56% (100) were aged 9-13 years and 77 (44%) were 14-19. 49% (86) had been orphaned by one parent, and 19% (33) by both. 59% (104) were severely underweight (BMI < 16). 47% presented with advanced HIV (WHO stage III/IV). 93% were virally supressed (< 250 copies/mL). 38 (21%) of ALHIV were on a second-line ART after first-line virological failure. Qualitative interviewing highlighted factors limiting adherence and the central role that HIV counsellors play for both ALHIV patients and caregivers. CONCLUSIONS: Our study shows good clinical, immunological, and virological outcomes for a cohort of Myanmar adolescents living with HIV, despite a majority being severely underweight, presenting with Stage III or IV illness, and the prevalence of comorbid infections (TB). Many treatment and adherence challenges were articulated in qualitative interviewing but emphasized the importance of actively engaging adolescents in their treatment. Comprehensive HIV care for this population must include routine viral load testing and social support programs.

Multicountry Analysis of Spectrum of Clinical Manifestations of Children <5 Years of Age Hospitalized with Diarrhea.

After introduction of rotavirus vaccine, other pathogens might become leading causes of hospitalizations for severe diarrhea among children <5 years of age. Our study in 33 hospitals in 7 countries found acute gastroenteritis accounted for most (84%) reported hospitalizations of children with diarrhea. Bloody and persistent diarrhea each accounted for <1%.

Routine Immediate Eye Examination at the Point of Care for Diagnosis of AIDS-Related Cytomegalovirus Retinitis Among Patients With a CD4 Count <100 in Myanmar.

A retrospective review of diagnosis of cytomegalovirus retinitis (CMVR) before and after introduction of routine immediate eye examination among AIDS patients in Myanmar with an absolute CD4 T-cell count <100 cells/µL demonstrated an increased detection of CMVR from 1.1% (14/1233) to 10.7% (65/608), an improvement of ~10-fold. Diagnosis of CMVR was achieved a mean of 2 days after clinic enrollment.

Global impact of rotavirus vaccine introduction on rotavirus hospitalisations among children under 5 years of age, 2008-16: findings from the Global Rotavirus Surveillance Network.

BACKGROUND: Rotavirus vaccine use in national immunisation programmes has led to declines in hospital admissions for rotavirus gastroenteritis among children; however, the global impact of rotavirus vaccine introduction has not been described using primary data. We describe the impact of rotavirus vaccine introduction on admissions for acute rotavirus gastroenteritis in primarily low-income and middle-income countries, using 9 years of data from the WHO-coordinated Global Rotavirus Surveillance Network (GRSN). METHODS: Between Jan 1, 2008, and Dec 31, 2016, children younger than 5 years of age who were admitted to hospital with acute gastroenteritis were prospectively enrolled in GRSN sites. We included sites that enrolled children and collected stool specimens monthly and tested at least 100 specimens annually in the impact analysis, with a separate analysis taking into account site continuity. We compared proportions of acute gastroenteritis cases positive for rotavirus in the pre-vaccine and post-vaccine periods and calculated mean proportion changes for WHO regions, with 95% CIs; these findings were then compared with interrupted time series analyses. We did further sensitivity analyses to account for rotavirus vaccination coverage levels and sites that collected specimens for at least 11 months per year and tested at least 80 specimens per year. We also analysed the age distribution of rotavirus-positive cases before and after vaccine introduction. FINDINGS: 403 140 children younger than 5 years of age admitted to hospital with acute gastroenteritis from 349 sites in 82 countries were enrolled over the study period, of whom 132 736 (32·9%) were positive for rotavirus. We included 305 789 children from 198 sites in 69 countries in the impact analysis. In countries that had not introduced rotavirus vaccine in their national immunisation programmes, rotavirus was detected in 38·0% (95% CI 4·8-73·4) of admissions for acute gastroenteritis annually whereas in those that have introduced the vaccine, rotavirus was detected in 23·0% (0·7-57·7) of admissions for acute gastroenteritis, showing a 39·6% (35·4-43·8) relative decline following introduction. Interrupted time series analyses confirmed these findings. Reductions by WHO regions ranged from 26·4% (15·0-37·8) in the Eastern Mediterranean Region to 55·2% (43·0-67·4) in the European Region and were sustained in nine countries (contributing up to 31 sites) for 6-10 years. The age distribution of children with rotavirus gastroenteritis shifted towards older children after rotavirus vaccine introduction. INTERPRETATION: A significant and sustained reduction in the proportion of hospital admissions for acute gastroenteritis due to rotavirus was seen among children younger than 5 years in GRSN sites following rotavirus vaccine introduction. These findings highlight the need to incorporate rotavirus vaccines into immunisation programmes in countries that have not yet introduced them and underline the importance of high-quality surveillance. FUNDING: The GRSN receives funding from Gavi, the Vaccine Alliance and the US Centers for Disease Control and Prevention. No specific funding was provided for this Article.

Characteristics of immunized and un-immunized students, including non-medical exemptions, in Ontario, Canada: 2016-2017 school year.

BACKGROUND: Our objectives were: (1) to quantify and describe un-immunized students in Ontario, Canada and assess the extent to which these students have exemptions; and (2) to quantify and describe students with non-medical exemptions (NMEs), including what proportion have up-to-date immunizations. METHODS: We examined Ontario students 7 to 17 years-of-age in the 2016-2017 school year using information within a centralized immunization repository. We identified and described students with different immunization/exemption classifications by age, sex, school type, geography and area-level material deprivation using descriptive and multivariable logistic regression analyses. Finally, we assessed the immunization status of students with NMEs, by antigen. RESULTS: We found that students could be recorded as un-immunized with or without an NME, or be immunized with an NME. From a cohort of 1.65 million students, 2.9% of students had zero vaccine doses recorded, and of these 68% had no exemption of any kind. A total of 2.4% of students had an NME. Of these, 39% were un-immunized and 61% had received ≥1 vaccine. Among all students with NMEs, 19-48% had up-to-date immunizations, varying by antigen. Factors associated with increased odds of having a NME and being un-immunized included: attendance at private and 'other' schools, rural residence, and geography. Older age and greater area-level deprivation were associated with a reduced odds. CONCLUSIONS: Our assessment revealed that Ontario students with NMEs cannot be assumed to be un-immunized and at risk for all vaccine-preventable diseases. Conversely, not all un-immunized students had NMEs suggesting that future studies of un-immunized children in Ontario must consider additional factors beyond NME status alone. Other jurisdictions that use NME data to inform research and surveillance of vaccine hesitancy and risks for VPD outbreaks may wish to undertake a similar assessment to determine how well student NMEs correlate with student immunization status.

Progress toward regional measles elimination - worldwide, 2000-2014.

In 2000, the United Nations General Assembly adopted the Millennium Development Goals (MDG), with MDG4 being a two-thirds reduction in child mortality by 2015, and with measles vaccination coverage being one of the three indicators of progress toward this goal.* In 2010, the World Health Assembly established three milestones for measles control by 2015: 1) increase routine coverage with the first dose of measles-containing vaccine (MCV1) for children aged 1 year to ≥90% nationally and ≥80% in every district; 2) reduce global annual measles incidence to fewer than five cases per million population; and 3) reduce global measles mortality by 95% from the 2000 estimate (1).† In 2012, the World Health Assembly endorsed the Global Vaccine Action Plan§ with the objective to eliminate measles in four World Health Organization (WHO) regions by 2015. WHO member states in all six WHO regions have adopted measles elimination goals. This report updates the 2000­2013 report (2) and describes progress toward global control and regional measles elimination during 2000­2014. During this period, annual reported measles incidence declined 73% worldwide, from 146 to 40 cases per million population, and annual estimated measles deaths declined 79%, from 546,800 to 114,900. However, progress toward the 2015 milestones and elimination goals has slowed markedly since 2010. To resume progress toward milestones and goals for measles elimination, a review of current strategies and challenges to improving program performance is needed, and countries and their partners need to raise the visibility of measles elimination, address barriers to measles vaccination, and make substantial and sustained additional investments in strengthening health systems.

Determining the Provincial and National Burden of Influenza-Associated Severe Acute Respiratory Illness in South Africa Using a Rapid Assessment Methodology.

Local disease burden data are necessary to set national influenza vaccination policy. In 2010 the population of South Africa was 50 million and the HIV prevalence was 11%. We used a previously developed methodology to determine severe influenza burden in South Africa. Hospitalized severe acute respiratory illness (SARI) incidence was calculated, stratified by HIV status, for four age groups using data from population-based surveillance in one site situated in Gauteng Province for 2009-2011. These rates were adjusted for each of the remaining 8 provinces based on their prevalence of risk factors for pneumonia and healthcare-seeking behavior. We estimated non-hospitalized influenza-associated SARI from healthcare utilization surveys at two sites and used the percent of SARI cases positive for influenza from sentinel surveillance to derive the influenza-associated SARI rate. We applied rates of hospitalized and non-hospitalized influenza-associated SARI to census data to calculate the national number of cases. The percent of SARI cases that tested positive for influenza ranged from 7-17% depending on age group, year, province and HIV status. In 2010, there were an estimated 21,555 total severe influenza cases in HIV-uninfected individuals and 13,876 in HIV-infected individuals. In 2011, there were an estimated 29,892 total severe influenza cases in HIV-uninfected individuals and 17,289 in HIV-infected individuals. The incidence of influenza-associated SARI was highest in children <5 years and was higher in HIV-infected than HIV-uninfected persons in all age groups. Influenza virus was associated with a substantial amount of severe disease, especially in young children and HIV-infected populations in South Africa.

Estimating costs of care for meningitis infections in low- and middle-income countries.

Meningitis infections are often associated with high mortality and risk of sequelae. The costs of treatment and care for meningitis are a great burden on health care systems, particularly in resource-limited settings. The objective of this study is to review data on the costs of care for meningitis in low- and middle-income countries, as well as to show how results could be extrapolated to countries without sound data. We conducted a systematic review of the literature from six databases to identify studies examining the cost of care in low- and middle-income countries for all age groups with suspected, probable, or confirmed meningitis. We extracted data on treatment costs and sequelae by infectious agent and/or pathogen, where possible. Using multiple regression analysis, a relationship between hospital costs and associated determinants was investigated in order to predict costs in countries with missing data. This relationship was used to predict treatment costs for all 144 low- and middle-income countries. The methodology of conducting a systematic review, extrapolating, and setting up a standard database can be used as a tool to inform cost-effectiveness analyses in situations where cost of care data are poor. Both acute and long-term costs of meningitis could be extrapolated to countries without reliable data. Although only bacterial causes of meningitis can be vaccine-preventable, a better understanding of the treatment costs for meningitis is crucial for low- and middle-income countries to assess the cost-effectiveness of proposed interventions in their country. This cost information will be important as inputs in future cost-effectiveness studies, particularly for vaccines.

Global invasive bacterial vaccine-preventable diseases surveillance--2008-2014.

Meningitis and pneumonia are leading causes of morbidity and mortality in children globally infected with Streptococcus pneumoniae (pneumococcus), Neisseria meningitidis, and Haemophilus influenzae causing a large proportion of disease. Vaccines are available to prevent many of the common types of these infections. S. pneumoniae was estimated to have caused 11% of deaths in children aged <5 years globally in the pre-pneumococcal conjugate vaccine (PCV) era. Since 2007, the World Health Organization (WHO) has recommended inclusion of PCV in childhood immunization programs worldwide, especially in countries with high child mortality. As of November 26, 2014, a total of 112 (58%) of all 194 WHO member states and 44 (58%) of the 76 member states ever eligible for support from Gavi, the Vaccine Alliance (Gavi), have introduced PCV. Invasive pneumococcal disease (IPD) surveillance that includes data on serotypes, along with meningitis and pneumonia syndromic surveillance, provides important data to guide decisions to introduce PCV and monitor its impact.

A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide.

DF2 (DRNFLRFamide), a FMRFamide-like peptide, has been shown to increase the amount of transmitter released at crayfish neuromuscular junctions. Here, we examined a possible role for the cyclic nucleotide monophosphates, cAMP and cGMP, in DF2's effects on synaptic transmission. The effects of DF2 on synaptic transmission were monitored by recording excitatory postsynaptic potentials (EPSPs) in the deep abdominal extensor muscles of the crayfish, Procambarus clarkii. A number of activators and inhibitors were used to determine whether or not cAMP, cGMP, protein kinase A (PKA) and protein kinase G (PKG) mediate the effect of this neuropeptide. Phosphodiesterase inhibitors, known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of DF2 on synaptic transmission. Activators of PKA (Sp-cAMPS) and PKG (8-pCPT-cGMP) increase EPSP amplitude, mimicking the effects of DF2. Inhibitors of PKA (Rp-cAMPS) and PKG (Rp-8-pCPT-cGMPS) each block a portion of the response to the peptide, and when applied together these two inhibitors completely block the response. Taken together, these results indicate that cyclic nucleotides and cyclic nucleotide-dependent protein kinases are necessary components of the pathway underlying modulation by this neuropeptide.

Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein.

Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.

Sorting of the respiratory syncytial virus matrix protein into detergent-resistant structures is dependent on cell-surface expression of the glycoproteins.

The interaction of the respiratory syncytial virus (RSV) Matrix (M) protein with the plasma membrane was investigated using polyclonal and monoclonal antisera raised against recombinant M expressed in bacteria. M bound mainly to the plasma membrane, although a significant proportion bound to internal membranes. However, no localisation of M with the Golgi was observed, suggesting that transport of M to the plasma membrane was independent of the transport mechanism for the viral glycoproteins. Expression from a recombinant baculovirus demonstrated the ability of M to bind membranes in the absence of viral glycoprotein expression. When cell-surface expression of the viral glycoproteins was prevented using Brefeldin A, M was still found in association with the plasma membrane, but the characteristics of M's membrane-binding ability were different to that found in untreated infected cells. In the presence of normal glycoprotein expression, M was sorted into lipid rafts and, in addition, formed structures that could only be disrupted by treatment with high salt buffers, a feature suggesting an interaction with the cytoskeleton or the formation of strong intramolecular associations. Brefeldin A prevented M from being sorted into lipid rafts or from forming strong intramolecular associations. Brefeldin A also affected the stability of M bound to the plasma membrane, as M was more readily dissociated in the presence of the inhibitor. Coexpression of M and F resulted in the incorporation of M into lipid rafts but did not cause the formation of the strong intramolecular bonds, suggesting that additional factors are required for this phenomena.