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Background The influenza activity of the 2019/20 season remained high and widespread in the United States with type B viruses predominating the early season. The majority of B viruses characterized belonged to B/Victoria (B/Vic) lineage and contained a triple deletion of amino acid (aa) 162–164 in hemagglutinin (3DEL). These 3DEL viruses are antigenically distinct from B/Colorado/06/2017 (CO/06)—the B/Vic vaccine component of the 2018/19 and 2019/20 seasons representing the viruses with a double deletion of aa 162–163 in hemagglutinin (2DEL). Methods We performed molecular characterization and phylogenetic analysis of circulating B/Vic viruses. We also conducted hemagglutination inhibition (HAI) assay using archived human postvaccination sera collected from healthy subjects administered with different types of 2018/19 or 2019/20 seasonal vaccines. Their HAI cross-reactivity to representative 3DEL viruses was analyzed. Results The CO/06-specific human postvaccination sera, after being adjusted for vaccine type, had significantly reduced HAI cross-reactivity toward representative 3DEL viruses, especially the 136E+150K subgroup. The geometric mean titers against 3DEL viruses containing 136E+150K mutations were 1.6-fold lower in all populations (P = .051) and 1.9-fold lower in adults (P = .016) compared with those against the 136E+150N viruses. Conclusions Our results indicate that postvaccination antibodies induced by the B/Vic vaccine component of the 2019/20 influenza season had reduced HAI cross-reactivity toward predominant 3DEL viruses in the United States. A close monitoring of the 3DEL 136E+150K subgroup is warranted should this subgroup return and predominate the 2020/21 influenza season.

Virions are a common antigen source for many viral vaccines. One limitation to using virions is that the antigen abundance is determined by the content of each protein in the virus. This caveat especially applies to viral-based influenza vaccines where the low abundance of the neuraminidase (NA) surface antigen remains a bottleneck for improving the NA antibody response. Our systematic analysis using recent H1N1 vaccine antigens demonstrates that the NA to hemagglutinin (HA) ratio in virions can be improved by exchanging the viral backbone internal genes, especially the segment encoding the polymerase PB1 subunit. The purified inactivated virions with higher NA content show a more spherical morphology, a shift in the balance between the HA receptor binding and NA receptor release functions, and induce a better NA inhibitory antibody response in mice. These results indicate that influenza viruses support a range of ratios for a given NA and HA pair which can be used to produce viral-based influenza vaccines with higher NA content that can elicit more balanced neutralizing antibody responses to NA and HA., Author summary Influenza vaccines are produced on a large scale to meet the annual U.S. and global demand. To efficiently produce the required number of influenza vaccine doses, virions are commonly used as the antigen source due to their high viral protein content. A draw-back to using virions is that the final antigen composition of the vaccine is determined by the inherent properties of the vaccine virus. While this approach for influenza vaccines is beneficial for the more abundant HA antigen, it likely limits the protective response generated by the less abundant NA antigen. Our results demonstrate that the NA and HA content in vaccine viruses can be optimized by changing the internal genes of the vaccine virus, thereby preserving the surface antigens. The increase in the virion NA content that was achieved elicited higher NA antibody titres and generated more balanced neutralizing antibody responses to HA and NA. Since HA and NA neutralizing antibodies are both protective, this approach could help to improve the suboptimal efficacy of current influenza vaccines and to generate vaccines that provide broader coverage against circulating strains.

Understanding the extent and limitation of viral genome evolution can provide insight about potential drug and vaccine targets. Influenza B Viruses (IBVs) infect humans in a seasonal manner and causes significant morbidity and mortality. IBVs are negative-sense single-stranded RNA viruses with a segmented genome and can be divided into two antigenically distinct lineages. The two lineages have been circulating and further evolving for almost four decades. The immune response to IBV infection can lead to antibodies that target the strain causing the infection. Some antibodies are cross-reactive and are able to bind strains from both lineages but, because of antigenic drift and immunodominance, both lineages continue to evolve and challenge human health. Here we investigate changes in the genomes of an IBVs from each lineage after passage in tissue culture in the presence of human sera containing polyclonal antibodies directed toward antigenically and temporally distinct viruses. Our previous analysis of the fourth segment, which encodes the major surface protein HA, revealed a pattern of change in which signature sequences from one lineage mutated to the signature sequences of the other lineage. Here we analyze genes from the other genomic segments and observe that most of the quasispecies’ heterogeneity occurs at the same loci in each lineage. The nature of the variants at these loci are investigated and possible reasons for this pattern are discussed. This work expands our understanding of the extent and limitations of genomic change in IBV.

Background Reduced seasonal influenza vaccine effectiveness (VE) was observed in individuals who received repeated annual vaccinations. Preexisting influenza antibody levels were also found inversely correlated with postvaccination titers. These reports suggest that preexisting immunity may affect contemporary seasonal vaccine performance. Methods Influenza A/H3 specific cross-reactivity of postvaccination sera from humans with or without preexisting immunity was assessed by hemagglutination inhibition (HAI) assay. Ferret antisera induced by repeated H3 exposures were also subjected to HAI, antibody affinity, and antibody avidity analyses. Results Human postvaccination sera derived from subjects with or without preexisting immunity showed different cross-reactivity against H3 variant viruses. Similarly, the breadth of cross-reactive ferret antibodies induced by repeated H3 exposures was also broadened. Antigenic differences between H3 viruses characterized by ferret antisera became smaller as the number of exposures increased. Although repeated H3 exposures induced "original antigenic sin" phenomena in HAI titers against later exposed viruses, resultant ferret antibodies showed gradually enhanced avidity for different H3/hemagglutinin. Increased antibody avidity was found to be inversely correlated with decreased antigenic differences among H3 viruses characterized. Conclusions Our results suggest that repeated H3 exposures imprinted not only antibody quantity but also antibody quality. The "naive" ferret model currently used for vaccine strain selection does not recapitulate the complexity of human preexisting immunity. Vaccine strains identified hereby may not provide coverage sufficient for those who were frequently infected and/or vaccinated, leading to the reduced VE observed.

The influenza virus NS1 protein interacts with a wide range of proteins to suppress the host cell immune response and facilitate virus replication. The amino acid sequence of the 2009 pandemic virus NS1 protein differed from sequences of earlier related viruses. The functional impact of these differences has not been fully defined. Therefore, we made mutations to the NS1 protein based on these sequence differences, and assessed the impact of these changes on host cell interferon (IFN) responses. We found that viruses with mutations at position 171 replicated efficiently but did not induce expression of interferon genes as effectively as wild-type viruses in A459 lung epithelial cells. The decreased ability of these NS1 mutant viruses to induce IFN gene and protein expression correlated with decreased activation of STAT1 and lower levels of IFN-stimulated gene (ISG) expression. These findings demonstrate that mutations at position 171 in the NS1 protein result in decreased expression of IFN and ISGs by A549 cells. Consequently, these viruses may be more virulent than the parental strains that do not contain mutations at position 171 in the NS1 protein.

Background Effectiveness of seasonal influenza vaccines mainly depends upon how well vaccine strains represent circulating viruses; mismatched strains can lead to reduced protection. Humans have complex influenza exposure histories that increase with age, which may lead to different postvaccination responses to emerging influenza variants. Recent observational studies also suggest that prior vaccination may influence the performance of current seasonal vaccines. Methods To elucidate the effects of age and influenza preexposures on cross-reactivity of vaccination-induced human antibodies, we generated antigenic maps based on postvaccination hemagglutination inhibition titers against representative H3 variants circulating during the 2015-2016, 2014-2015, and 2012-2013 influenza seasons. Results Antigenic maps determined using sera from subjects 18-64 and ≥65 years of age correlated well with each other but poorly with those determined using sera from children. Antigenic maps derived from human postvaccination sera with H1 influenza preexposure also correlated poorly with those derived from sera with neither H1 nor type B influenza preexposure, and the correlation lessened considerably over time. In contrast, antigenic maps derived from human postvaccination sera with only type B influenza preexposure consistently showed good correlation with those derived from sera with neither H1 nor type B influenza preexposure. Conclusions Our results suggest an age-specific difference in human postvaccination responses. Our findings also suggest that prior exposure to H1 or type B influenza may differentially affect cross-reactivity of vaccination-induced H3-specific hemagglutination inhibition antibody responses, and consequently might affect vaccine effectiveness. Our study highlights the need to study the impact of prior exposure on influenza vaccine performance.

Made available in DSpace on 2019-10-04T12:41:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-07-02 National Health and Medical Research Council of Australia Background-The uptake of proven stroke treatments varies widely. We aimed to determine the association of evidence-based processes of care for acute ischemic stroke (AIS) and clinical outcome of patients who participated in the HEADPOST (Head Positioning in Acute Stroke Trial), a multicenter cluster crossover trial of lying flat versus sitting up, head positioning in acute stroke. Methods and Results-Use of 8 AIS processes of care were considered: reperfusion therapy in eligible patients; acute stroke unit care; antihypertensive, antiplatelet, statin, and anticoagulation for atrial fibrillation; dysphagia assessment; and physiotherapist review. Hierarchical, mixed, logistic regression models were performed to determine associations with good outcome (modified Rankin Scale scores 0-2) at 90 days, adjusted for patient and hospital variables. Among 9485 patients with AIS, implementation of all processes of care in eligible patients, or defect-free care, was associated with improved outcome (odds ratio, 1.40; 95% CI, 1.18-1.65) and better survival (odds ratio, 2.23; 95% CI, 1.62-3.09). Defect-free stroke care was also significantly associated with excellent outcome (modified Rankin Scale score 0-1) (odds ratio, 1.22; 95% CI, 1.04-1.43). No hospital characteristic was independently predictive of outcome. Only 1445 (15%) of eligible patients with AIS received all processes of care, with significant regional variations in overall and individual rates. Conclusions-Use of evidence-based care is associated with improved clinical outcome in AIS. Strategies are required to address regional variation in the use of proven AIS treatments. Univ New South Wales, Fac Med, George Inst Global Hlth, Sydney, NSW, Australia Ctr Estudios Clin, Inst Ciencias & Innovac Med, Santiago, Chile Clin Alemana Univ Desarrollo, Fac Med, Serv Neurol, Dept Neurol & Psiquiatria, Santiago, Chile Peking Univ, Hlth Sci Ctr, George Inst Global Hlth, Beijing, Peoples R China Australian Catholic Univ, Nursing Res Inst, St Vincents Hlth Australia Sydney, Sydney, NSW, Australia Univ Cent Lancashire, Fac Hlth & Wellbeing, Preston, Lancs, England Univ Chile, Fac Med, Dept Ciencias Neurol, Santiago, Chile Univ Sao Paulo, Ribeirao Preto Med Sch, Stroke Serv Neurol Div, Ribeirao Preto, Brazil Fukuoka Univ, Fac Med, Dept Prevent Med & Publ Hlth, Fukuoka, Fukuoka, Japan Univ Leicester, Dept Cardiovasc Sci, Leicester Biomed Res Ctr, Leicester, Leics, England Univ Leicester, Natl Inst Hlth Res, Leicester Biomed Res Ctr, Leicester, Leics, England Univ Edinburgh, Ctr Clin Brain Sci, Edinburgh, Midlothian, Scotland George Inst Global Hlth, Sydney, NSW, Australia Univ Desarrollo, Serv Neurol, Dept Neurol & Psiquiatria Clin Alemana Santiago, Santiago, Chile St Vincents Hlth Australia Sydney, Sydney, NSW, Australia Australian Catholic Univ, Sydney, NSW, Australia Univ Cent Lancashire, Stroke Practice Res Unit, Sch Hlth, Lancashire Clin Trials Unit, Preston, Lancs, England Univ Leicester, Dept Cardiovasc Sci, Cardiovasc Res Ctr, British Heart Fdn, Leicester, Leics, England Univ Kelaniya, Dept Pharmacol, Fac Med, Colombo, Sri Lanka Christian Med Coll & Hosp, Dept Neurol, Ludhiana, India Kaohsiung Med Univ & Hosp, Dept Neurol, Kaohsiung, Taiwan Linkou Chang Gung Mem Hosp, Dept Neurol, Taipei, Taiwan Peking Union Med Coll Hosp, Beijing, Peoples R China Univ Sao Paulo, Ribeirao Preto Sch Med, Ribeirao Preto, Brazil Monash Univ, Dept Epidemiol & Prevent Med, Melbourne, Vic, Australia Univ Cent Lancashire, Sch Hlth Sci, Coll Hlth & Wellbeing, Preston, Lancs, England Univ Desarrollo, Santiago, Chile Univ New South Wales, Neurosci Res Australia, Sydney, NSW, Australia Natl Univ Singapore, Dept Pharmacol, Singapore, Singapore Bradford Royal Infirm, Bradford Inst Hlth Res, Leeds, W Yorkshire, England Christian Med College, Ludhiana, Punjab, India RemediumOne Pvt Ltd, Kandy, Sri Lanka Kaohsiung Med Univ, Chung Ho Mem Hosp, Kaohsiung, Taiwan Calvary Publ Hosp Bruce, Bruce, Australia Royal North Shore Hosp, St Leonards, NSW, Australia Royal Prince Alfred Hosp, Camperdown, NSW, Australia Concord Repatriat Gen Hosp, Camperdown, NSW, Australia Fiona Stanley Hosp, Camperdown, NSW, Australia Macquarie Base Hosp, Melbourne, Vic, Australia Univ Sao Paulo, Fac Med Ribeirao Preto, Hosp Clin, Sao Paulo, Brazil Hosp Governador Celso Ramos, Florianopolis, SC, Brazil Sao Paulo State Univ, Hosp Fac Med Botucatu, UNESP, Sao Paulo, Brazil Yangquan Coalmine Grp Gen Hosp, Yangquan, Peoples R China Nanjing Med Univ, Nanjing Hosp 1, Nanjing, Jiangsu, Peoples R China 85 Hosp Peoples Liberat Army, Beijing, Peoples R China Chifeng Univ, Affiliated Hosp, Chifeng, Peoples R China Beijing Pinggu Hosp, Beijing, Peoples R China Wenzhou Med Univ, Affiliated Hosp 1, Wenzhou, Peoples R China Soochow Univ, Affiliated Hosp 2, Suzhou, Peoples R China Hebei Med Univ, Hosp 3, Shijiazhuang, Hebei, Peoples R China Capital Med Univ, Xuanwu Hosp, Beijing, Peoples R China Jining Med Univ, Affiliated Hosp, Jining, Peoples R China Third Peoples Hosp Huizhou, Huizhou, Peoples R China Second Cangzhou Cent Hosp, Cangzhou, Peoples R China Shanghai Jiao Tong Univ, Hosp Affiliated, Sch Med, Shanghai, Peoples R China Baogang Hosp, Baotou, Peoples R China Harbin Med Univ, Affiliated Hosp 1, Harbin, Heilongjiang, Peoples R China Shanghai Jiao Tong Univ Sch, Tong Ren Hosp, Peoples Hosp Hejian City, Shanghai, Peoples R China Peking Univ, Shougang Hosp, Beijing, Peoples R China Guangzhou Med Univ, Affiliated Hosp 3, Guangzhou, Guangdong, Peoples R China Second Hosp Nanchang, Nanchang, Jiangxi, Peoples R China Bethune Int Peace Hosp, Beijing, Peoples R China Hua Henan Prov Peoples Hosp, Beijing, Peoples R China Hua Henan Prov Peoples Hosp, Zhengzhou, Henan, Peoples R China Shanghai Jiao Tong Univ, Sch Med, Shanghai Ninth Peoples Hosp, Shanghai, Peoples R China Shijiazhuang Cent Hosp, Shijiazhuang, Hebei, Peoples R China Hosp Base San Jos de Osorno, Osorno, Chile Christian Med Coll & Hosp, Ludhiana, Punjab, India Unit Narayana Hrudayalaya Ltd, Mazumdar Shaw Med Ctr, Bangalore, Karnataka, India Dr Ramesh Cardiac & Multispecial Hosp Pvt Ltd, Guntur, India Post Grad Inst Med Educ & Res, Chandigarh, India Baby Mem Hosp Ltd, Calicut, Kerala, India Sree Chitra Tirunal Inst Med Sci & Technol, Thiruvananthapuram, Kerala, India Sri Jayawardenepura Gen Hosp, Sri Jayawardenepura Kott, Sri Lanka Colombo North Teaching Hosp, Colombo, Sri Lanka Colombo South Coching Hosp, Colombo, Sri Lanka Linkou Chang Gung Mem Hosp, Taoyuan, Taiwan Taipei Med Univ, Shuang Ho Hosp, Taipei, Taiwan En Chu Kong Hosp, New Taipei, Taiwan Natl Cheng Kung Univ Hosp, Tainan, Taiwan Queen Alexandra Hosp, Portsmouth, Hants, England Portsmouth Hosp NHS Natl Hlth Serv iTrust, Portsmouth, Hants, England Univ Coll London Hosp NHS Fdn Trust, London, England Univ Hosp Birmingham NHS Fdn Trust, Queen Elizabeth Hosp Birmingham, Birmingham, W Midlands, England Southend Univ Hosp NHS Fdn Trust, Southend On Sea, England Kings Coll Hosp London, London, England York Teaching Hosp NHS Fdn Trust, York, N Yorkshire, England Nottingham Univ Hosp NHS Trust, Nottingham, England Blackpool Teaching Hosp NHS Fdn Trust, Blackpool, England Gloucestershire Hosp NHS Fdn Trust, Gloucestershire Royal Hosp, Gloucester, England Teaching Hospi NHS Fdn Trust, London, England Royal Cornwall Hosp NHS Trust, Truro, England Royal London Hosp, Barts Hlth NHS Trust, London, England Sheffield Teaching Hosp NHS Fdn Trust, Sheffield, S Yorkshire, England Cambridge Univ Hosp NHS Fdn Trust, Addenbrookes Hosp, Cambridge, England Royal Victoria Hosp, Belfast Hlth & Social Care Trust, London, England Pinderfields Gen Hosp, Mid Yorkshire Hosp NHS Trust, Wakefield, England Royal Devon & Exeter NHS Fdn Trust, London, England Bradford Teaching Hosp NHS Fdn Trust, Bradford, W Yorkshire, England Great Western Hosp NHS Fdn Trust, Swindon, Wilts, England Watford Dist Gen Hosp, Watford, England Imperial Coll Healthcare NHS Trust, London, England Medway NHS Fdn Trust, Gillingham, England Royal Liverpool & Broadgreen Univ NHS Trust, Liverpool, Merseyside, England Calderdale & Huddersfield NHS Fdn Trust, Huddersfield, W Yorkshire, England Hywel Dda Univ Hlth Board, Haverfordwest, Wales Chesterfield Royal Hosp NHS Fdn Trust, Calow, England Luton & Dunstable Univ Hosp NHS Fdn Trust, Luton, Beds, England Countess Chester Hosp NHS Fdn Trust, Chester, Cheshire, England Peterborough City Hosp, Peterborough, Cambs, England Univ Hosp Leicester NHS Trust, Leicester Royal Infirm, Leicester, Leics, England Dist Hosp NHS Fdn Trust, London, England Barnsley Hosp NHS Fdn Trust, Barnsley, England Dorset Cty Hosp NHS Fdn Trust, Dorchester, England Royal Bournemouth & Christchurch Hosp NHS Fdn Tru, Royal Bournemouth Hosp, Bournemouth, Dorset, England Southern Hlth & Social Care Trust, Craigavon Area Hosp, Portadown, England United Lincolnshire Hosp NHS Trust, Pilgrim Hosp, Lincoln, England Bedford Hosp NHS Trust, Bedford, England Sao Paulo State Univ, Hosp Fac Med Botucatu, UNESP, Sao Paulo, Brazil National Health and Medical Research Council of Australia: 1066966

A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines.

Background The potency of inactivated influenza vaccines is determined using a single‐radial immunodiffusion (SRID) assay and requires standardized reagents consisting of a Reference Antigen and an influenza strain‐specific antiserum. Timely availability of reagents is a critical step in influenza vaccine production, and the need for backup approaches for reagent preparation is an important component of pandemic preparedness. Objectives When novel H7N9 viruses emerged in China in 2013, candidate inactivated H7N9 influenza vaccines were developed for evaluation in clinical trials, and reagents were needed to measure vaccine potency. Methods We previously described an alternative approach for generating strain‐specific potency antisera, utilizing modified vaccinia virus Ankara vectors to produce influenza hemagglutinin (HA)‐containing virus‐like particles (VLPs) for immunization. Vector‐produced HA antigen is not dependent upon the success of the traditional bromelain‐digestion and HA purification. Results Antiserum for H7N9 vaccines, produced after immunization of sheep with preparations of bromelain‐HA (br‐HA), was not optimal for the SRID assay, and the supply of antiserum was limited. However, antiserum obtained from sheep boosted with VLPs containing H7 HA greatly improved the ring quality in the SRID assay. Importantly, this antiserum worked well with both egg‐ and cell‐derived antigen and was distributed to vaccine manufacturers. Conclusions Utilizing a previously developed approach for preparing vaccine potency antiserum, we have addressed a major bottleneck encountered in preparation of H7N9 vaccine reagents. The combination of br‐HA and mammalian VLPs for sequential immunization represents the first use of an alternative approach for producing an influenza vaccine potency antiserum.

This novel platform can detect and differentiate different influenza subtypes from a single sample., Conventional methods for detection and discrimination of influenza viruses are time consuming and labor intensive. We developed a diagnostic platform for simultaneous identification and characterization of influenza viruses that uses a combination of nanomicroarray for screening and multiplex next-generation sequencing (NGS) assays for laboratory confirmation. The nanomicroarray was developed to target hemagglutinin, neuraminidase, and matrix genes to identify influenza A and B viruses. PCR amplicons synthesized by using an adapted universal primer for all 8 gene segments of 9 influenza A subtypes were detected in the nanomicroarray and confirmed by the NGS assays. This platform can simultaneously detect and differentiate multiple influenza A subtypes in a single sample. Use of these methods as part of a new diagnostic algorithm for detection and confirmation of influenza infections may provide ongoing public health benefits by assisting with future epidemiologic studies and improving preparedness for potential influenza pandemics.

Seasonal influenza vaccine formulas change almost every year yet information about how this affects the antibody repertoire of vaccine recipients is inadequate. New vaccine virus strains are selected, replacing older strains to better match the currently circulating strains. But even while the vaccine is being manufactured the circulating strains can evolve. The ideal response to a seasonal vaccine would maintain antibodies toward existing strains that might continue to circulate, and to generate cross-reactive antibodies, particularly towards conserved influenza epitopes, potentially limiting infections caused by newly evolving strains. Here we use the hemagglutination inhibition assay to analyze the antibody repertoire in subjects vaccinated two years in a row with either identical vaccine virus strains or with differing vaccine virus strains. The data indicates that changing the vaccine formulation results in an antibody repertoire that is better able to react with strains emerging after the vaccine virus strains are selected. The effect is observed for both influenza A and B strains in groups of subjects vaccinated in three different seasons. Analyses include stratification by age and sex.

In February the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winter in the Northern Hemisphere. These recommendations are based on data collected by National Influenza Centres (NICs) through the WHO Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations of the 2013–2014 Northern Hemisphere influenza vaccine composition.

Hemagglutinin (HA) is the major antigen in influenza vaccines and glycosylation is known to influence its antigenicity. Embryonated hen eggs are traditionally used for influenza vaccine production, but vaccines produced in mammalian and insect cells were recently licensed. This raises the concern that vaccines produced with different cell systems might not be equivalent due to differences in their glycosylation patterns. Thus, we developed an analytical method to monitor vaccine glycosylation through a combination of nanoLC/MSE and quantitative MALDI-TOF MS permethylation profiling. We then used this method to examine glycosylation of HA’s from two different influenza H5N1 strains produced in five different platforms, including hen eggs, three different insect cell lines (High Five™, expresSF+® and glycoengineered expresSF+), and a human cell line (HEK293). Our results demonstrated that (1) sequon utilization is not necessarily equivalent in different cell types, (2) there are quantitative and qualitative differences in the overall N-glycosylation patterns and structures produced by different cell types, (3) ~20% of the N-glycans on the HAs produced by High Five™ cells are core α1,3-fucosylated structures, which may be allergenic in humans, and (4) our method can be used to monitor differences in glycosylation during the cellular glycoengineering stages of vaccine development.

Influenza virus infection has a significant impact on public health, since it is a major cause of morbidity and mortality. It is not well-known whether influenza virus infection affects cell death and human immunodeficiency virus (HIV)-1 replication in HIV-1-infected patients. Using a lymphoma cell line, Jurkat, we examined the in vitro effects of pandemic influenza A (H1N1) virus (pH1N1) infection on cell death and HIV-1 RNA production in infected cells. We found that pH1N1 infection increased apoptotic cell death through Fas and Bax-mediated pathways in HIV-1-infected Jurkat cells. Infection with pH1N1 virus could promote HIV-1 RNA production by activating host transcription factors including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB), nuclear factor of activated T-cells (NFAT) and activator protein 1 (AP-1) through mitogen-activated protein kinases (MAPK) pathways and T-cell antigen receptor (TCR)-related pathways. The replication of HIV-1 latent infection could be reactivated by pH1N1 infection through TCR and apoptotic pathways. These data indicate that HIV-1 replication can be activated by pH1N1 virus in HIV-1-infected cells resulting in induction of cell death through apoptotic pathways.

In February and September each year the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winters in the Northern and Southern Hemispheres respectively. These recommendations are based on data collected by National Influenza Centres (NIC) through the Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations for the 2012 Southern Hemisphere influenza vaccine composition.

Highly pathogenic H5N1 avian influenza viruses emerged in 1996 and have since evolved so extensively that a single strain can no longer be used as a prepandemic vaccine or diagnostic reagent. We therefore sought to identify the H5N1 strains that may best serve as cross-reactive diagnostic reagents. We compared the cross-reactivity of 27 viruses of clades 0, 1, 2.1, 2.2, 2.3, and 4 and of four computationally designed ancestral H5N1 strains by hemagglutination inhibition (HI) and microneutralization (MN) assays. Antigenic cartography was used to analyze the large quantity of resulting data. Cartographs of HI titers with chicken red blood cells were similar to those of MN titers, but HI with horse red blood cells decreased antigenic distances among the H5N1 strains studied. Thus, HI with horse red blood cells seems to be the assay of choice for H5N1 diagnostics. Whereas clade 2.2 antigens were able to detect antibodies raised to most of the tested H5N1 viruses (and clade 2.2-specific antisera detected most of the H5N1 antigens), ancestral strain A exhibited the widest reactivity pattern and hence was the best candidate diagnostic reagent for broad detection of H5N1 strains., link_to_OA_fulltext

Wild type human influenza viruses do not usually grow well in embryonated hens' eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

The 2009 H1N1 pandemic highlights the need to better understand influenza A infectivity and antigenicity. Relative to other recent seasonal H1N1 influenza strains, the 2009 H1N1 virus grew less efficiently in eggs, which hindered efforts to rapidly supply vaccine. Using lentiviral pseudotypes bearing influenza hemagglutinin (HA-pseudotypes) we evaluated a glutamine to arginine mutation at position 223 (Q223R) and glycosylation at residue 276 in HA for their effects on infectivity and neutralization. Q223R emerged during propagation in eggs and lies in the receptor binding site. We found that the Q223R mutation greatly enhanced infectivity of HA-pseudotypes in human cells, which was further augmented by inclusion of the viral neuraminidase (NA) and M2 proteins. Loss of glycosylation at residue 276 did not alter infectivity. None of these modifications affected neutralization. These findings provide information for increasing 2009 H1N1HA-pseudotype titers without altering antigenicity and offer insights into receptor use.

Pseudotype reporter viruses are being used as safe, quantitative, and high-throughput tools for assessing antibody neutralization for many viruses, including influenza. However, characterization of pseudotypes containing influenza hemagglutinin (HA-pseudotypes) is needed before this system is widely adopted for evaluating neutralizing antibodies in sera following vaccination or infection. In this report HA-pseudotype stocks were analyzed for HA content, stability, and performance in neutralization assays under various conditions. HA-pseudotypes produced with HA genes of H5 strains representing clades 1, 2.2, and 2.3.4 consistently contain similar HA contents, and infectivity was not greatly affected by the purity of the HA-pseudotype preparations or variations in storage conditions. HA-pseudotype neutralization titers using a reference serum panel were also consistent across a wide range of dilutions of HA-pseudotype stocks and correlated well with results from microneutralization assays involving replicating influenza. Concentrated HA-pseudotypes were further shown to work well in hemagglutination inhibition assays. Finally, antisera elicited by genetically modified HA, with changes in the polybasic cleavage site that have been used in some H5 vaccines and reduce pathogenicity, gave identical neutralization titers against HA-pseudotypes with wild type or modified HA. These findings support continued development of HA-pseudotypes as a robust tool for analyzing sera in vaccine and serologic studies.

Influenza vaccines form an important component of the global response against infections and subsequent illness caused in man by influenza viruses. Twice a year, in February and September, the World Health Organisation through its Global Influenza Surveillance Network (GISN), recommends appropriate influenza viruses to be included in the seasonal influenza vaccine for the upcoming Northern and Southern Hemisphere winters. This recommendation is based on the latest data generated from many sources and the availability of viruses that are suitable for vaccine manufacture. This article gives a summary of the data and background to the recommendations for the 2009-2010 Northern Hemisphere influenza vaccine formulation.

The M gene segment of influenza A virus has been shown to be a contributing factor to the high growth phenotype. However, it remains largely unknown why matrix protein 1 (M1), the major structural protein encoded by M gene, exhibits pH-dependent conformational changes during virus replication. Understanding the mechanisms underlying efficient virus replication can help to develop strategies not only to combat influenza infections but also to improve vaccine supplies. M(NLS-88R) and M(NLS-88E) are two M1 mutants differing by only a single amino acid: G88R vs G88E. G88R but not G88E was the compensatory mutation naturally selected by the virus after its nuclear localization signal was disrupted. Our study shows that, compared with M(NLS-88E) M1, M(NLS-88R) M1 dissociated quickly from viral ribonucleoproteins (vRNPs) at higher pH and took less time to dissemble in vitro, despite forming thicker matrix layer and having stronger association with vRNP in assembled virions. Correspondingly, M(NLS-88R) replicated more efficiently and was genetically more stable than M(NLS-88E). Crystallographic analysis indicated that M(NLS-88R) M1, like wild-type M1, is able to switch from a face-to-back-oriented conformation to a face-to-face-oriented conformation when pH drops from neutral to acidic, whereas G88E mutation causes M(NLS-88E) M1 to be trapped in a face-to-face-arranged conformation regardless of environmental pH. Our results suggest that maintaining M1 pH-dependent conformational flexibility is critical for efficient virus replication, and position 88 is a key residue controlling M1 pH-dependent conformational changes. Our findings provide insights into developing M1-based antiviral agents.

The preparation of high-yield prepandemic influenza virus H5N1 strains has presented a challenge to both researchers and vaccine manufacturers. The reasons for the relatively low yield of the H5N1 strains are not fully understood, but it might be partially dependent on the interactions between the hemagglutinin (HA) or neuraminidase (NA) surface glycoprotein and other influenza virus proteins. In this study, we have constructed chimeras between the A/Puerto Rico/8/34 (PR8) NA gene and the A/Vietnam/1203/2004 (VN1203) NA gene that have resulted in an increase in the virus yield of the reassortant viruses without a significant loss of NA activity. By combining the amino terminus of NA from the PR8 strain with the carboxy terminus of NA from VN1203, the surface epitopes unique to the H5N1 VN1203 NA glycoprotein are maintained. This reassortant virus had a higher titer and total protein yield in eggs, grew to a higher titer, produced large plaques on MDCK cells, and retained NA activity. This work describes a novel recombinant technique designed to increase the yields of vaccine candidates for the production of pandemic influenza virus vaccines. The relationship between the infectivity and protein yield of the reassortants also is discussed.

Pseudotype reporter viruses provide a safe, quantitative, and high-throughput tool for assessing antibody neutralization for many viruses, including high pathogenicity H5 and H7 influenza A strains. However, adapting this system to other influenza subtypes has been hampered by variations in the protease cleavage site of hemagglutinin (HA) that make it less susceptible to the cleavage required for infectivity. In this report several proteases, reporter vectors, and cell substrates were evaluated while optimizing pseudovirus production, and robust methods were established for sensitive and specific neutralization of pseudotypes carrying influenza H1, H3, and H5 subtype HA that correlates well with titers obtained in microneutralization assays involving replicating influenza virus These findings should facilitate broad use of HA-pseudotypes that remove the need for infectious virus in a range of applications, including neutralization assays for serological surveys of viral infection and evaluations of vaccine sera.

The poor performance of 2014–15 Northern Hemisphere (NH) influenza vaccines was attributed to mismatched H3N2 component with circulating epidemic strains. Using human serum samples collected from 2009–10, 2010–11 and 2014–15 NH influenza vaccine trials, we assessed their cross-reactive hemagglutination inhibition (HAI) antibody responses against recent H3 epidemic isolates. All three populations (children, adults and older adults) vaccinated with the 2014–15 NH egg- or cell-based vaccine, showed >50% reduction in HAI post-vaccination geometric mean titers against epidemic H3 isolates from those against egg-grown H3 vaccine strain A/Texas/50/2012 (TX/12e). The 2014–15 NH vaccines, regardless of production type, failed to further extend HAI cross-reactivity against H3 epidemic strains from previous seasonal vaccines. Head-to-head comparison between ferret and human antisera derived antigenic maps revealed different antigenic patterns among representative egg- and cell-grown H3 viruses characterized. Molecular modeling indicated that the mutations of epidemic H3 strains were mainly located in antibody-binding sites A and B as compared with TX/12e. To improve vaccine strain selection, human serologic testing on vaccination-induced cross-reactivity need be emphasized along with virus antigenic characterization by ferret model.

Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine (121XP, X-179A and X-181) viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys226Glu) was located in the Ca antigenic site of HA (present in 18% of the population). Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T (Glu252Asp, 47%) in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks.

We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process—from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.

The matrix protein (M1) of influenza virus plays an essential role in viral replication. Our previous studies have shown that basic amino acids 101RKLKR105 of M1 are involved in RNP binding and nuclear localization. For the present work, the functions of 101RKLKR105 were studied by introducing mutations into the M gene of influenza virus A/WSN/33 by reverse genetic methods. Individual substitution, R101S or R105S, had a minimal effect on viral replication. In contrast, the double mutation R101S-R105S was synergistic and resulted in temperature sensitivity reflected by reduced viral replication at a restrictive temperature. To investigate the in vivo effect on infection, BALB/c mice were infected with either A/WSN/33 wild-type (Wt) or mutant viruses and assessed for signs of illness, viral replication in the lungs, and survival rates. The results from mouse studies indicated that the R101S-R105S double mutant virus was strongly attenuated, while single mutant viruses R101S and R105S were minimally attenuated compared to A/WSN33 Wt under the same conditions. In challenge studies, mice immunized by infection with R101S-R105S were fully protected from lethal challenge with A/WSN/33. The replication and attenuating properties of R101S-R105S suggest its potential in development of live influenza virus vaccines.

Our previous studies with influenza A viruses indicated that the association of M1 with viral RNA and nucleoprotein (NP) is required for the efficient formation of helical ribonucleoprotein (RNP) and for the nuclear export of RNPs. RNA-binding domains of M1 map to the following two independent regions: a zinc finger motif at amino acid positions 148 to 162 and a series of basic amino acids (RKLKR) at amino acid positions 101 to 105. Altering the zinc finger motif of M1 reduces viral growth slightly. A substitution of Ser for Arg at either position 101 or position 105 of the RKLKR domain partially reduces the nuclear export of RNP and viral replication. To further understand the role of the zinc finger motif and the RKLKR domain in viral assembly and replication, we introduced multiple mutations by using reverse genetics to modify these regions of the M gene of influenza virus A/WSN/33. Of multiple mutants analyzed, a double mutant, R101S-R105S, of RKLKR resulted in a temperature-sensitive phenotype. The R101S-R105S double mutant had a greatly reduced ratio of M1 to NP in viral particles and a weaker binding of M1 to RNPs. These results suggest that mutations can be introduced into the RKLKR domain to control viral replication.

The hemagglutination inhibition assay (HAI) is widely used to evaluate vaccine-induced antibody responses as well as to antigenically characterize influenza viruses. The results of an HAI assay are based on an endpoint titration where the titers are generally manually interpreted and recorded by a well-trained expert. For serological applications, the lack of standardization in endpoint interpretation and interference from non-specific inhibitors in clinical samples can translate into a high degree of variability in the results. For example, tilting HAI plates at 45-60 degrees to look for a "tear drop pattern" with avian red blood cells is a common practice by many, but not all, research laboratories. In this work, we tested the hypothesis that an automated image analysis algorithm can be used to achieve an accurate and non-subjective interpretation of HAI assays-specifically without the need to tilt plates. In a side-by-side comparison study performed during FDA's biannual serological screening process for influenza viruses, titer calls for more than 2200 serum samples were made by the Cypher One automated hemagglutination analyzer without tilting and by an expert human with tilting. The comparison yielded 95.6% agreement between the expert reader and automated interpretation method (within ± 1 dilution) for the complete dataset. Performance was also evaluated relative to the type of red blood cell (turkey and guinea pig) and influenza strain (12 different viruses). For the subset that utilized guinea pig red blood cells (~44% of the samples), for which no plate tilting was required, the agreement with an expert reader was 97.2%. For the subset that utilized turkey red blood cells (~56% of the samples), for which plate tilting was necessary by the expert reader, the agreement was 94.3%. Overall these results support the postulate that algorithm-based interpretation of a digital record with no plate tilting could replace manual reading for greater consistency in HAI assays.

Amplification of influenza A virus gene segments by reverse transcription-polymerase chain reaction (RT-PCR) can be combined with enzymatic digestion to reveal unique restriction fragment length polymorphisms specific for H1N1 and H3N2 subtype viruses. We have used the method to provide a rapid, specific and reproducible identification of the genotype of high-growth influenza reassortants derived from A/Puerto Rico/8/34 (PR8). Digestion of the gene segments amplified from wild-type viruses, PR8 and reassortants at sites unique to either the wild-type strain or to PR8 provided positive, unambiguous identification of the origin of each of the internal genes, and distinguished the internal genes of both H1N1 and H3N2 strains from those of PR8. This method has also permitted us to quickly confirm that reassorting has occurred and to optimize the selection of reassortant clones with maximum number of PR8 internal genes. Since the method can detect 1-10% of a second strain in a mixed population, the method can also be used to detect samples containing more than one viral subtype and to assess the purity of influenza viruses used for manufacturing vaccines.

A HeLa T4 cell line containing a defective human immunodeficiency virus type 1 (HIV-1) DNA (HD4) was isolated. After transactivation with Tat, the HD4 DNA was transcribed into a single 3.7-kb mRNA that encodes a chimeric CD4/Env protein and a multitarget-ribozyme directed against multiple sites within the gp120 coding region of HIV-1 RNA (Chen et al., 1992). Early steps in HIV infection such as entry, reverse transcription, and proviral DNA formation were not affected in HD4 cells, and HD4 was efficiently transactivated after either HIV-1 or HIV-2 infections. HIV-2, which lacks all of the HIV-1-specific ribozyme target sites, replicated to high levels in HD4 cells whereas HIV-1 replication was selectively inhibited. Despite a reduced accumulation of all HIV-1 transcripts, transactivation of HD4 was efficient. Surprisingly, the most abundant, multiply spliced mRNAs were reduced even though they lack all of the ribozyme target sites. These results strongly suggest that the ribozyme co-localizes with unspliced HIV-1 pre-mRNA and/or genomic HIV-1 RNA in the nucleus. Cleavage of these precursor RNAs explains the reduction of all spliced and unspliced HIV-1 RNAs. Cleavage of genomic RNA probably contributed to the three-fold reduction in the infectivity of viral progeny. Thus, the HD4 ribozyme RNA functioned as a ribozyme in the nucleus and as a mRNA for a chimeric CD4/Env protein in the cytoplasm. Its unusual large size for a ribozyme (3.7 kb) indicates that, in the future, other antiviral proteins, like negative transdominant mutant HIV-1 proteins, may also be encoded to increase its antiviral potential in a gene therapy approach.

It is not well-known whether apoptosis signaling affects influenza virus infection and reproduction in human lung epithelial cells. Using A549 cell line, we studied the relationship of some apoptosis-associated molecules with novel pandemic influenza A (H1N1) virus, A/California/04/2009. Infected cells displayed upregulated Fas ligand, activated FADD and caspase-8, and downregulated FLIP in the extrinsic apoptotic pathway. p53 expression increased and Bcl-XL expression decreased in the intrinsic pathway. Expression of pre-apoptotic molecules (FasL, FADD, and p53) increased virus replication, while inhibition of activity of FADD, caspase-8 and caspase-3, and expression of anti-apoptotic proteins (FLIP and Bcl-XL) decreased virus replication. p38, ERK and JNK from MAPK pathways were activated in infected cells, and inhibition with their inhibitors diminished virus replication. In the p38 superfamily, p38α expression increased viral RNA production, while expression of p38β and p38γ decreased. These data indicated that influenza virus induces apoptotic signaling pathways, which benefit virus replication.

We previously developed a rapid and simple gold nanoparticle(NP)-based genomic microarray assay for identification of the avian H5N1 virus and its discrimination from other influenza A virus strains (H1N1, H3N2). In this study, we expanded the platform to detect the 2009 swine-origin influenza A virus (H1N1/2009). Multiple specific capture and intermediate oligonucleotides were designed for the matrix (M), hemagglutinin (HA), and neuraminidase (NA) genes of the H1N1/2009 virus. The H1N1/2009 microarrays were printed in the same format as those of the seasonal influenza H1N1 and H3N2 for the HA, NA, and M genes. Viral RNA was tested using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining. The signal from the 4 capture-target-intermediates of the HA and NA genes was specific for H1N1/2009 virus and showed no cross hybridization with viral RNA from other influenza strains H1N1, H3N2, and H5N1. All of the 3 M gene captures showed strong affinity with H1N1/2009 viral RNA, with 2 out of the 3 M gene captures showing cross hybridization with the H1N1, H3N2, and H5N1 samples tested. The current assay was able to detect H1N1/2009 and distinguish it from other influenza A viruses. This new method may be useful for simultaneous detection and subtyping of influenza A viruses and can be rapidly modified to detect other emerging influenza strains in public health settings.

The glycoproteins, heamagglutinin (HA) and neuraminidase (NA) of influenza virus confer host protective immune responses during vaccination, which is the most effective approach for preventing influenza-associated morbidity and mortality. Since the functional balance between the HA and NA proteins may affect viral receptor binding and replication, a pandemic influenza A virus (H1N1 pdm09), strain A/Texas/05/2009, was optimized to elevate its HA antigen content by modifying the NA gene. In this study, we have constructed two 2:6 reassortant viruses between pdmH1N1 (A/Texas/05/2009) and A/Puerto Rico/8/34 (PR8), in which the NA gene of A/Texas/05/2009 was modified to contain part of the NA gene from PR8. One chimeric NA virus has the PR8 transmembrane (TM) region (HNtm 2:6) and the other contains both the PR8 NA TM and stem regions (HNst 2:6). Using quantitative reverse phase-HPLC (RP-HPLC) analysis, we observed that the HNst2:6 virus contains a higher HA1 content than HN2:6 wild type. In addition, this mutant virus displays a higher HA1 to nucleoprotein (NP) ratio, based on gel electrophoresis densitometry analysis. Furthermore, the neuraminidase activity of purified HNst 2:6 virus is approximately 30% lower than that of HN2:6 virus, which is suggestive of a lower incorporation of NA into the viral envelope. Therefore, we propose that the reduction of NA packaging in the virion may lead to a compensatory increase of HA. Such an improvement in HA yield is possibly beneficial to H1N1 pdm09 vaccine production.

Background Influenza A virus has been detected in the blood of some infected individuals, and may pose a safety concern for collection, handling and transport of specimens for epidemiological and public health investigations if infectious virus is present in samples. Furthermore the effect of storage on virus stability and infectivity has not been well studied. Methods We examined the stability of novel pandemic influenza A (H1N1) virus RNA when the virus was stored in phosphate buffered saline (PBS), plasma, or buffy coated blood at either room temperature or 4°C using a sensitive Taqman RT-PCR assay. We also investigated virus infectivity using the EID50 assay when virus was stored in PBS, plasma, or buffy coats isolated from blood at 4°C. Results Viral RNA stability was affected by the matrix used for storage. The recovery of viral RNA was highest when virus was stored in PBS with lower amounts being recovered from plasma and buffy coats at either room temperature or 4°C. Incubation time did not appear to be a major factor for viral RNA stability, although there was gradual decline after longer periods post-incubation. Both sample matrix and incubation time affected virus infectivity. The decay in virus infectivity was greatest in PBS followed by buffy coats and plasma. Virus infectivity was abolished in buffy coats at day 20 post-incubation when virus concentrations were low. Conclusion These data indicate that encapsidated viral RNA was stable overall in all three liquid matrices at room temperature or 4°C although it was most stable in PBS; virus infectivity in buffy coats at 4°C decayed in a time dependent manner while it remained unchanged in plasma. These findings have implications for storage, handling and transport of blood derived samples from influenza patients for epidemiological and laboratory investigations. It should be noted that there is little known about influenza viremia, and whether influenza viruses can be transmitted by blood or blood derived samples.

Pandemic 2009 H1N1 influenza A virus (2009 H1N1) differs from H1N1 strains that circulated in the past 50 years, but resembles the A/New Jersey/1976 H1N1 strain used in the 1976 swine influenza vaccine. We investigated whether sera from persons immunized with the 1976 swine influenza or recent seasonal influenza vaccines, or both, neutralize 2009 H1N1. Using retroviral pseudovirions bearing hemagglutinins on their surface (HA-pseudotypes), we found that 77% of the sera collected in 1976 after immunization with the A/New Jersey/1976 H1N1 swine influenza vaccine neutralized 2009 H1N1. Forty five percent also neutralized A/New Caledonia/20/1999 H1N1, a strain used in seasonal influenza vaccines during the 2000/01–2006/07 seasons. Among adults aged 48–64 who received the swine influenza vaccine in 1976 and recent seasonal influenza vaccines during the 2004/05–2008/09 seasons, 83% had sera that neutralized 2009 H1N1. However, 68% of age-matched subjects who received the same seasonal influenza vaccines, but did not receive the 1976 swine influenza vaccine, also had sera that neutralized 2009 H1N1. Sera from both 1976 and contemporary cohorts frequently had cross-neutralizing antibodies to 2009 H1N1 and A/New Caledonia/20/1999 that mapped to hemagglutinin subunit 2 (HA2). A conservative mutation in HA2 corresponding to a residue in the A/Solomon Islands/3/2006 and A/Brisbane/59/2007 H1N1 strains that circulated in the 2006/07 and 2007/08 influenza seasons, respectively, abrogated this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in HA2 and suggest that HA2 may be evolving under direct or indirect immune pressure., Author Summary Influenza A viruses mutate to escape neutralization by antibodies. These mutations predominantly occur in the globular head of the hemagglutinin protein, while the stalk is more conserved. Pandemic 2009 H1N1 influenza virus differs from seasonal H1N1 strains that circulated in the past 50 years and resembles a strain that did not circulate but was used in the 1976 swine influenza vaccine. We investigated whether persons immunized with either the 1976 swine influenza or recent seasonal influenza vaccines, or both, have antibodies that cross-neutralize pandemic 2009 H1N1. Sera from 1976 swine influenza vaccine trials cross-neutralized pandemic 2009 H1N1 and to a lesser extent the A/New Caledonia/20/1999 H1N1 strain that was used in vaccines during the 2000/01–2006/07 influenza seasons. Sera from persons who received several seasonal influenza vaccines containing A/New Caledonia/20/1999 H1N1 cross-neutralized pandemic 2009 H1N1, regardless of whether they received the 1976 swine influenza vaccine. We found that cross-neutralization between 2009 H1N1 and A/New Caledonia/20/1999 frequently mapped to the hemagglutinin stalk. A mutation in the stalk of strains circulating during the 2007/08–2008/09 seasons abrogates this neutralization. These findings highlight a cross-neutralization determinant influenced by a point mutation in the hemagglutinin stalk and suggest that the stalk may be evolving under direct or indirect immune pressure.

Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection.

The variation of highly pathogenic avian influenza H5N1 virus results in gradually increased virulence in poultry, and human cases continue to accumulate. The neuraminidase (NA) stalk region of influenza virus varies considerably and may associate with its virulence. The NA stalk region of all N1 subtype influenza A viruses can be divided into six different stalk-motifs, H5N1/2004-like (NA-wt), WSN-like, H5N1/97-like, PR/8-like, H7N1/99-like and H5N1/96-like. The NA-wt is a special NA stalk-motif which was first observed in H5N1 influenza virus in 2000, with a 20-amino acid deletion in the 49(th) to 68(th) positions of the stalk region. Here we show that there is a gradual increase of the special NA stalk-motif in H5N1 isolates from 2000 to 2007, and notably, the special stalk-motif is observed in all 173 H5N1 human isolates from 2004 to 2007. The recombinant H5N1 virus with the special stalk-motif possesses the highest virulence and pathogenicity in chicken and mice, while the recombinant viruses with the other stalk-motifs display attenuated phenotype. This indicates that the special stalk-motif has contributed to the high virulence and pathogenicity of H5N1 isolates since 2000. The gradually increasing emergence of the special NA stalk-motif in H5N1 isolates, especially in human isolates, deserves attention by all.

BackgroundEfficient targeted gene transfer and cell type specific transgene expression are important for the safe and effective expression of transgenesin vivo. Enveloped viral vectors allow insertion of exogenous membrane proteins into their envelopes, which could potentially aid in the targeted transduction of specific cell types. Our goal was to specifically target cells that express the T cell tropic HIV-1 envelope protein (Env) using the highly specific interaction of Env with its cellular receptor (CD4) inserted into the envelope of an HIV-1-based viral vector.ResultsTo generate HIV-1-based vectors carrying the CD4 molecule in their envelope, the CD4 ectodomain was fused to diverse membrane anchors and inserted together with the HIV-1 coreceptor CXCR4 into the envelopes of HIV-1 vector particles. Independent of the type of CD4 anchor, all chimeric CD4 proteins inserted into HIV-1 vector envelopes and the resultant HIV(CD4/CXCR4) particles were able to selectively confer neomycin resistance to cells expressing the fusogenic T cell tropic HIV-1 Env protein. Unexpectedly, in the absence of Env on the target cells, all vector particles carrying the CD4 ectodomain anchored in their envelope adhered to various cell types without infecting these cells. This cell adhesion was very avid. It was independent of the presence of Env on the target cell, the type of CD4 anchor or the presence of CXCR4 on the particle. In mixed cell populations with defined ratios of Env+/Env-cells, the targeted transduction of Env+cells by HIV(CD4/CXCR4) particles was diminished in proportion to the number of Env-cells.ConclusionVector diversion caused by a strong, non-selective cell binding of CD4+-vector particles effectively prevents the targeted transduction of HIV-1 Env expressing cells in mixed cell populations. This Env-independent cell adhesion severely limits the effective use of targeted HIV(CD4/CXCR4) vectors designed to interfere with HIV-1 replicationin vivo. Importantly, the existence of this newly described and remarkably strong CD4-dependent cell adhesion suggests that the multiple viral efforts to reduce CD4 cell surface expression may, in part, be to prevent cell adhesion to non-target cells and thereby to increase the infectivity of viral progeny. Preventing CD4 down-modulation by HIV-1 might be an effective component of a multi-faceted antiviral strategy.

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.

The campaign of 2009–2010 Northern Hemisphere seasonal vaccination was concurrent with the 2009 H1N1 pandemic. Using a hemagglutination inhibition (HAI) assay, we evaluated the immunogenicity and cross-reactivity of 2009–2010 inactivated trivalent influenza vaccine (TIV) in US adult and elderly populations. Vaccination of TIV resulted in a robust boost on the antibody response of all subjects to seasonal A/Brisbane/59/2007 (H1N1) and A/Uruguay/716/2007 (H3N2) with over 70% of recipients reaching a seroprotective titer of 40. B/Brisbane/60/2008 was the least immunogenic among the three seasonal vaccine strains with