A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity. Keywords: influenza virus; hemagglutinin; receptor; sialyllactose-N-tetraose; gel-capture assay.

Identifying crucial gaps in our knowledge of the life-history of avian influenza viruses - An Australian perspective

We review our current knowledge of the epidemiology and ecology of avian influenza viruses (AIVs) in Australia in relation to the ecology of their hosts. Understanding the transmission and maintenance of low-pathogenic avian influenza (LPAI) viruses deserves scientific scrutiny because some of these may evolve to a high-pathogenic AIV (HPAI) phenotype. That the HPAI H5N1 has not been detected in Australia is thought to be a result of the low level of migratory connectivity between Asia and Australia. Some AIV strains are endemic to Australia, with Australian birds acting as a reservoir for these viruses. However, given the phylogenetic relationships between Australian and Eurasian strains, both avian migrants and resident birds within the continent must play a role in the ecology and epidemiology of AIVs in Australia. The extent to which individual variation in susceptibility to infection, previous infections, and behavioural changes in response to infection determine AIV epidemiology is little understood. Prevalence of AIVs among Australian avifauna is apparently low but, given their specific ecology and Australian conditions, prevalence may be higher in little-researched species and under specific environmental conditions.

Development and validation of the influenza intensity and impact questionnaire (FluiiQ™)

Clinical trials of new agents to reduce the severity and impact of influenza require accurate assessment of the effect of influenza infection. Because there are limited high-quality adult influenza Patient Reported Outcomes (PRO) measures, the aim was to develop and validate a simple but comprehensive questionnaire for epidemiological research and clinical trials.

Functional analysis of the leading malaria vaccine candidate AMA-1 reveals an essential role for the cytoplasmic domain in the invasion process

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Functional analysis of Plasmodium falciparum merozoite antigens : implications for erythrocyte invasion and vaccine development

Malaria is a major human health problem and is responsible for over 2 million deaths per year. It is caused by a number of species of the genus Plasmodium, and Plasmodium falciparum is the causative agent of the most lethal form. Consequently, the development of a vaccine against this parasite is a priority. There are a number of stages of the parasite life cycle that are being targeted for the development of vaccines. Important candidate antigens include proteins on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to manipulate the genome of Plasmodium species has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. This has provided new information on the role of merozoite antigens in erythrocyte invasion and also allows new approaches to address their potential as vaccine candidates.

Pandemic (H1N1) 2009 influenza in Australia : absenteeism and redeployment of emergency medicine and nursing staff

Objective: The aim of the present study was to examine the impact of Pandemic (H1N1) 2009 Influenza on the Australian emergency nursing and medicine workforce, specifically absenteeism and deployment.

Methods: Data were collected using an online survey of 618 members of the three professional emergency medicine or emergency nursing colleges.

Results: Despite significant increases in emergency demand during the Pandemic (H1N1) 2009 Influenza, 56.6% of emergency nursing and medicine staff reported absenteeism of at least 1 day and only 8.5% of staff were redeployed. Staff illness with influenza-like illness was reported by 37% of respondents, and 87% of respondents who became ill were not tested for the Pandemic (H1N1) 2009 Influenza. Of the respondents who became ill, 43% (n = 79) reported missing no days of work and only 8% of respondents (n = 14) reported being absent for more than 5 days. The mean number of days away from work was 3.73 (standard deviation = 3.63). Factors anecdotally associated with staff absenteeism (caregiver responsibilities, concern about personal illness, concern about exposing family members to illness, school closures, risk of quarantine, stress and increased workload) appeared to be of little or no relevance. Redeployment was reported by 8% of respondents and the majority of redeployment was for operational reasons.

Conclusion: Future research related to absenteeism, redeployment during actual pandemic events is urgently needed. Workforce data collection should be an integral part of organizational pandemic planning.

Effects of infection-induced migration delays on the epidemiology of avian influenza in wild mallard populations

Wild waterfowl populations form a natural reservoir of Avian Influenza (AI) virus, and fears exist that these birds may contribute to an AI pandemic by spreading the virus along their migratory flyways. Observational studies suggest that individuals infected with AI virus may delay departure from migratory staging sites. Here, we explore the epidemiological dynamics of avian influenza virus in a migrating mallard (Anas platyrhynchos) population with a specific view to understanding the role of infection-induced migration delays on the spread of virus strains of differing transmissibility. We develop a host-pathogen model that combines the transmission dynamics of influenza with the migration, reproduction and mortality of the host bird species. Our modeling predicts that delayed migration of individuals influences both the timing and size of outbreaks of AI virus. We find that (1) delayed migration leads to a lower total number of cases of infection each year than in the absence of migration delay, (2) when the transmission rate of a strain is high, the outbreak starts at the staging sites at which birds arrive in the early part of the fall migration, (3) when the transmission rate is low, infection predominantly occurs later in the season, which is further delayed when there is a migration delay. As such, the rise of more virulent AI strains in waterfowl could lead to a higher prevalence of infection later in the year, which could change the exposure risk for farmed poultry. A sensitivity analysis shows the importance of generation time and loss of immunity for the effect of migration delays. Thus, we demonstrate, in contrast to many current transmission risk models solely using empirical information on bird movements to assess the potential for transmission, that a consideration of infection-induced delays is critical to understanding the dynamics of AI infection along the entire flyway.

Host behaviour and physiology underpin individual variation in avian influenza virus infection in migratory Bewick's swans

Individual variation in infection modulates both the dynamics of pathogens and their impact on host populations. It is therefore crucial to identify differential patterns of infection and understand the mechanisms responsible. Yet our understanding of infection heterogeneity in wildlife is limited, even for important zoonotic host-pathogen systems, owing to the intractability of host status prior to infection. Using novel applications of stable isotope ecology and eco-immunology, we distinguish antecedent behavioural and physiological traits associated with avian influenza virus (AIV) infection in free-living Bewick's swans (Cygnus columbianus bewickii). Swans infected with AIV exhibited higher serum δ13C (-25.3 ± 0.4) than their non-infected counterparts (-26.3±0.2). Thus, individuals preferentially foraging in aquatic rather than terrestrial habitats experienced a higher risk of infection, suggesting that the abiotic requirements of AIV give rise to heterogeneity in pathogen exposure. Juveniles were more likely to be infected (30.8% compared with 11.3% for adults), shed approximately 15-fold higher quantity of virus and exhibited a lower specific immune response than adults. Together, these results demonstrate the potential for heterogeneity in infection to have a profound influence on the dynamics of pathogens, with concomitant impacts on host habitat selection and fitness.

Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Development of combinatorial RNAi transgenes targeting influenza virus

This research investigated the optimization of RNA interference against influenza A viruses. Results obtained in this study increase knowledge of the use of RNA interference in the context of creating antiviral transgenes capable of simultaneously targeting multiple viral genes and preventing the risk of viral escape.

Pandemic (H1N1) Influenza 2009 and Australian emergency departments : implications for policy, practice and pandemic preparedness

Objective: To describe the reported impact of Pandemic (H1N1) 2009 on EDs, so as to inform future pandemic policy, planning and response management.

Methods: This study comprised an issue and theme analysis of publicly accessible literature, data from jurisdictional health departments, and data obtained from two electronic surveys of ED directors and ED staff. The issues identified formed the basis of policy analysis and evaluation.

Results: Pandemic (H1N1) 2009 had a significant impact on EDs with presentation for patients with ‘influenza-like illness’ up to three times that of the same time in previous years. Staff reported a range of issues, including poor awareness of pandemic plans, patient and family aggression, chaotic information flow to themselves and the public, heightened stress related to increased workloads and lower levels of staffing due to illness, family care duties and redeployment of staff to flu clinics. Staff identified considerable discomfort associated with prolonged times wearing personal protective equipment. Staff believed that the care of non-flu patients was compromised during the pandemic as a result of overwork, distraction from core business and the difficulties associated with accommodating infectious patients in an environment that was not conducive.

Conclusions: This paper describes the breadth of the impact of pandemics on ED operations. It identifies a need to address a range of industrial, management and procedural issues. In particular, there is a need for a single authoritative source of information, the re-engineering of EDs to accommodate infectious patients and organizational changes to enable rapid deployment of alternative sources of care.