International Conference on Influenza August 24-26, 2015 West Drayton, London, UK

Wen-Chi Su is an Assistant Professor, Graduate Institute of Clinical Medical Science and also China Medical University Assistant Researcher, Research center for emerging viruses, China Medical University Hospital.

As viral proteins usually hijack or collaborate with the cellular proteins for executing their functions, it becomes important to identify host factors indispensable for influenza virus replication. Previously, we have conducted a novel genome-wide pooled shRNA screen to search for host factors important for influenza A virus (IAV) replication and have acquired some candidate genes. Here, we first confirmed that ribosomal RNA processing 1 homolog B (RRP1B), one of the candidates, is crucial for IAV replication. Moreover, mini-replicon assay as well as primer-extension assays showed that silencing RRP1B hampered viral RNA-dependent RNA polymerase (RdRp) activity. Furthermore, we showed that RRP1B binds to PB1 and PB2 of the RdRp as well as cellular mRNA. Silencing RRP1B reduced IAV mRNA levels in the presence of cycloheximide and inhibited RdRp complex binding to capped mRNA. Taken together, our study indicates that RRP1B is an important component of the IAV transcription complex and reveal new insights into the mechanism of IAV virus-host interaction.

Anna J.X. Zhang is a Research Assistant Professor belongs to the department of Microbiology at The University of Hong Kong.

Background
Novel avian H7N9 influenza virus infects humans causing severe respiratory diseases which lead to a case mortality higher than 30%. Genomic analysis showed that these H7N9 viruses contained relative low frequency of T cell epitopes on their HA protein compared to other subtypes of influenza viruses, suggesting that these viruses have low immunogenicity. In this study, serum antibody responses to H7N9 infection in BALB/c mice were investigated.
Methods
Groups of 6-8 weeks old female BALB/c mice were infected with 103, 104 or 105 PFU of H7N9 human isolate, A/Anhui/01/2013 (AH1) and A/ZheJiang/DTID-ZJU01/2013. For comparison, 2009 pandemic H1N1 virus (A/HK/415742/09), and a recombinant virus rg-PR8-H7-N9 which contains HA and NA gene from ZJ1 virus and the 6 internal genes from A/PR/8/34 (H1N1) were also studied. At 7, 14 or 28 days after virus infection, serum samples from infected mice were examined for hemagglutination inhibition (HI) antibody and microneutralization (MN) antibody.
Results
After infection with AH1, all the mice developed serum hemagglutination inhibition (HI) antibody (GMT= 40-72.5) at day 14 post infection, the titer increased further at day 28 p.i. (GMT=171.5-201.6). However, most of the infected mice had no detectable neutralizing antibody even at day 28 p.i. by standard microneutralization (MN) assay. To study whether the lack of serum neutralizing antibody was also occurred in other H7N9 virus infection, 105 PFU of ZJ1 were inoculated to BALB/c mice, similar pattern of HI response was observed, but no detectable MN antibody. From these mouse anti-sera, low level of neutralizing activity could be detected by fluorescent focus microneutralization (FFMN) assay, but passive transfer of the anti-sera to infected mice showed no protection effects. Through comparison, we found that the pattern of antibody response to H7N9 infection is dramatically different from that of 2009 pandemic H1N1 virus infection, A/HK/415742/09 H1N1 induced robust high titer of HI and MN antibody responses at day 14 p.i., and the anti-sera protected mice from lethal infection when passive transferred to the same strain of virus infected mice. To study whether the observed antibody response in H7N9 infection is solely related to the proposed low frequency of T cell epitopes on its surface HA proteins, a recombinant influenza rg-PR8-H7-N9 virus was studied in mice. The results showed that rg-PR8-H7-N9 infection induced significantly stronger serum antibody responses. MN antibody titer reached GMT of 121.3 at day 14 p.i, which further increased to GMT of 278.5 at day 28 p. i.. The MN titers were significantly higher than that induced by wild type H7N9 virus, ZJ1. ZJ1 infected mice only produced very low MN antibody at day 28 p.i, with 2/10 mice had titer of 1:40, 7/10 mice had a titer of 1:20 (GMT = 20). These results suggest that the internal genes in the H7N9 virus may also affect the antibody responses.

Rene Kizek is a Professor and Head of Laboratory of Metallomics and Nanotechnology, Mendel University in Brno and Vice-Head of Research Group Leader of Submicron Systems and Nanodevices in Central European Institute of Technology. His research is mainly focused on effects of metal ions in organisms and their roles in various pathological processes mainly tumour diseases. Further, his team is aimed at developing new types of nanomaterials and testing these materials for nanomedical and nanomedicine purposes. He is an author of more than three hundred ISI indexed papers with more than 7000 citations and H-index 45.

Characterization of protein moieties can reveal important properties which can determine their biological fate from the perspective of their detection or possible interaction with binding partners. For our study, we employed substantial domain of influenza surface antigen hemagglutinin (HA)-sialic acid binding region. The N-terminal region of GP1 of Ebola which is critical for receptor binding referred to as the receptor-binding domain was employed for this study too. The aim of this study was to determine binding interaction of each of fragments with paramagnetic particles (PMPs). For analysis it is required their resuspension in PBS buffer pH 7 (phosphate buffered saline) at a ratio of 40 mg PMPs and 1 ml PBS. After six washing steps with PBS of pH 7 and a volume of 250 µl using an external magnetic field, one of the six synthesized peptides (ZEV 1-6) was added to each type of prepared modified super paramagnetic particles (MAN 99, MAN 105, MAN 123 and MAN 128) and incubated. Then, the particles were separated with a magnet, the solution was pipetted off and the particles were again washed three times with PBS, pH 7.0. The sample was concentrated by nitrogen evaporation Ultravap RC at 60o C. The dried sample was resuspended in 250 µl of dilution buffer in the sodium cycle. Finally, the binding capacity of the individual peptide fragments Ebola glycoprotein (ZEV 1-6) with the certain type of modified particles was determined.

Mihaela Lazar is a Researcher and main player in the National Surveillance Program of ILI (Influenza-like Illness) and SARI (Severe Acute Respiratory Infections) network in Romania.

In Romania, the influenza activity in 2014-2015 was characterized by co-circulation of influenza A(H3N2), A(H1N1)pdm09 and B viruses. Primary objective of our study was to monitor the emergence of HA variants of influenza virus in patients with influenza-like illness (ILI) and severe acute respiratory infections (SARI) selected from sentinel and non-sentinel systems by antigenic and genetic characterization. One hundred eleven strains were isolated on cell cultures. All A(H1N1)pdm09 viruses were antigenically characterized as A/California/7/2009 but a higher heterogeneity was observed for subtype H3N2 and type B indicating a mismatch between circulating and vaccine strains. Full hemagglutinin gene was sequenced from 22 original specimens (including an influenza B and H1N1 co-infection), from which 14 collected from patients hospitalized with SARI. Four of them had fatal evolution, although one had been vaccinated. All characterized viruses fell into clade A/California/7/2009, subgroup 6B, A/South Africa/3626/2013. Seventeen viruses type B fell into clade 3 (B/Phuket/3073/2013), four from vaccinated patients and three deaths. For A(H3N2) viruses, the complexity of genetic data interpretation must take into account the nature and location of the main mutations. The 22 sequenced strains H3N2 clustered into three different clades, 3C.2a and 3C.3a and 3C.3b. However, among deaths with laboratory-confirmed influenza, only 15% were caused by the H3N2 subtype versus 62.5% subtype H1N1 and 22.5% type B respectively. Our study emphasize the importance of monitoring viral evolution and the role of genetic data on understanding the virus-host interaction and disease severity as well as in selecting seasonal influenza vaccine’s strains.

Lubna Kouser graduated with a Bachelor’s degree in Biomedical Science from Brunel University in September 2011. Currently, a final year PhD student of Dr Kishore, in Brunel University, mainly focusing on the interaction of human properdin with Mycobacteria and influenza A virus. She has acquired various techniques such as gene cloning, PCR, qPCR, cDNA synthesis, recombinant protein expression in E. coli (globular heads A, B, C, TSRs, SPD, SPA) and baculovirus proteins, immunoassay, Western blots, purification of serum complement proteins: C1q, factor H, properdin, familiar with CL3 facilities and standard operating procedures, microscopy, purification and titration of Influenza A Virus.

Soluble molecules of the innate immune system play a major role in the initial defences against infection by pathogens. Properdin is a multimeric soluble protein released by activated neutrophils. It is critical in the stabilisation of the complement alternative pathway C3 convertases. Properdin is able to bind to certain pathogen surfaces, stimulating convertase formation and opsonisation. Our objective was to screen a range of purified human soluble factors involved in innate immunity and complement regulation for their capacity to inhibit haemagglutination of guinea pig red blood cells by human influenza A viruses in a complement-independent manner. Human soluble factors were purified from human serum by affinity chromatography. Alternatively, recombinant proteins were expressed in E.coli. Purified proteins were dialised and purity, molecular weight and appropriate folding were verified.
We found that purified properdin inhibits haemagglutination of all influenza A virus strains tested (A/Hong Kong/99 and A/Udorn/72 H3N2 viruses and A/WSN/33, A/Hong Kong/98 and pandemic A/UK/09 H1N1 viruses). Inhibition was more potent on H1N1 viruses including pH1N1. We found that properdin interacts with native viruses and purified haemagglutinin and neuraminidase envelope proteins. Quantitative real-time-PCR studies on M1 viral gene at early and late times post-infection shown that properdin down regulates infection of A549 pneumocytes by pH1N1 virus. Our findings reveal that human properdin binds influenza A virus and restricts infection of target cells. The observation that properdin interacts with influenza A virus without involving C3b is novel and worth further investigation.

Stefan has completed a degree in biology from University of Balearic Islands and a Master’s degree in Advanced Microbiology recognised by both University of Barcelona and University of Balearic Islands (Spain). He then enrolled for a PhD at Brunel University London and has learnt state of the art virology and cellular biology approaches, which has enabled him to study the molecular mechanism responsible for restriction of influenza viruses by human annexin A6.

Elucidation of networks of interactions between viruses and host cells will pave the way towards the understanding of molecular determinants of cytopathogenicity and ultimately the design of novel therapeutics.
Our strategy is to identify human restricting or enhancing factors of IAV infection. We specifically looked for interactions of cytosolic domains of IAV proteins with human factors using a yeast-two-hybrid screening approach. We found that the C-terminal domain of the highly pathogenic avian H5N1 virus M2 protein interacts with human annexin A6. We further confirmed the interaction of annexin A6 with the M2 protein of A/WSN/33 (H1N1) virus. Our initial study demonstrated that human annexin A6 restricts IAV infection in vitro (Ma et al., J Virol 2012). Interestingly, restriction was confirmed on all IAV strains tested. Using transmission electron microscopy, we found that annexin A6 expression alters IAV morphogenesis and restricts release of progeny virions.
Our current investigations aim at understanding the molecular mechanism responsible for IAV restriction by annexin A6.

Tai-Ling Chao is a PhD student studied in Dr. Sui-Yuan Chang 's lab in the department of clinical laboratory science medical biotechnology of National Taiwan University. The major study topics of Dr. Chang's laboratory are molecular virology of HIV and influenza virus. The first imported H7N9 case in Taiwan was identified and investigated in our lab, and the result was published on Clinical Infectious Disease in 2014. (Clin Infect Dis. (2014) 58 (2):242-246.)

An outbreak of a novel avian-origin reassortant H7N9 virus in China has raised great concerns due to the rapidly progressive lower respiratory tract infections in infected individuals in 2013. In this study, H7N9 viruses were isolated from the Day 0420, Day 0422, Day 0425 sputum and Day 0425 throat swab of the first imported case in Taiwan. Hem-Agglutinin (HA), neuraminidase and Matrix (M) full-length genome sequences were determined by using PacBio RS II platform. Two mutations, D19G and K330E, in the HA sequence were found in Day 0425 sputum and were speculated to have higher binding ability to human receptor compared with the wild type HA through calculating protein binding affinity. Half of the analyzed NA sequences amplified from four specimens have R289K, the oseltamivir-resistant mutation. Other oseltamivir-resistant mutations such as E115V and I219R were also identified. None of the amino acid mutations were detected in the M gene in all specimens. In conclusion, the population with higher human receptor binding affinity and drug resistance were identified after high dose oseltamivir treatment and might become the major group.

Sherwin Morgan completed his respiratory care training from Malcolm X College of Respiratory Care in Chicago, IL. He is an advanced respiratory care practitioner with the National Board for Respiratory Care in the United States. He is Clinical Practice and Development /Educator/Research Coordinator for the Department of Respiratory Care Services, Section of Pulmonary and Critical Care Medicine at the University of Chicago Medicine. He has published more than 25 peer review papers in multiple medical journals. He has designed, engineered, and collaborated with a number of research studies with the pulmonary medicine department.

Heliox, a helium-oxygen gas mixture, has been used for many decades to treat obstructive pulmonary disease. The lower density and higher viscosity of heliox relative to nitrogen-oxygen mixtures can significantly reduce airway resistance when an anatomic or partial air-flow obstruction is present. Respiratory infections caused by coronavirus / rhinovirus-enterovirus range from the common cold to severe acute respiratory distress syndrome (SARS). In infants, respiratory syndromes can cause bronchitis, viral bronchiolitis, and pneumonia in variable combinations and may produce enough air-flow obstruction to cause respiratory failure. These viral strains are now documented to come from multiple viral agents and continue to mutate into new strains. I present the case of a 10-month old Hispanic male treated with high flow nasal cannula and heliox to avoid intubation. Before heliox the patient was tachypnea 60 – 70 breaths/min and would desaturate to 84% as measured by pulse oximetry with suprasternal chest retractions. After heliox 70/30, his respiratory rate fell 31 to 38 breaths/min. Nutritional support was implemented on day 1 of heliox administration. He was maintained on heliox either 60/40 or 70/30 for 48 hours and heliox discontinued on day three. He was discharge from PICU on day 10 and discharged from the hospital 7 days later. The benefit of heliox itself appeared to be immediate and served as a bridge to support the patient while time and pharmacologic measures took effect and an underlying infection abated.

I graduated as biologist, specialization in biochemistry in 2009 at Maria Curie-Skłodowska University in Lublin. I completed the postgraduate studies in 2012 as Research Project Management and Commercialization of Research Results at University of Technology in Łódź. Since 2009 I have associated with Institute of Biochemistry and Biophysics Polish Academy of Sciences (Warsaw, Poland) where I worked as biologist in project concerning veterinary DNA vaccine against influenza and now I continue working in this interesting area as a PhD student.

Injection of plasmid or linear form of DNA encoding vaccine antigen can lead to the induction of immune response. This approach offers a number of potential advantages over traditional approaches, including the stimulation of both humoral and cell-mediated responses, the relative ease of large-scale manufacture and improves vaccine stability enabling withdrawal from restrictive and very expensive cold chain transport. The most important disadvantage is low level of antigen expression in-vivo and hence low immunogenicity of vaccine. Several strategies can be used to overcome this difficulty.

Our studies are focused on DNA vaccine against H5N1. To improve vaccine immunogenicity and reduce its cost of production we evaluated different strategies of modification of basic vaccine variant prepared. Basal H5 HA DNA vaccine was modified in the following ways: i - proteolytic cleavage site was removed, ii – codon usage was change using three different algorithm, iii – expression cassette was modified to permit DNA transport to nucleus increasing gene expression level, iv – RNA-OUT vector was used allowing selection without antibiotics and enabling in vivo expressed proteins transport to different cell compartments: endosome or secrete outside the cell. All vaccine variants were tested in mice and vaccine efficacy was measured by ELISA assay. Our results showed that tested doses of DNA vaccine elicit HA- dependent humoral response. The level of immunological response depended on the tested vaccine variant and the best modification was chosen for further studies.

This work was supported by Innovative Economy Program, No. WND-POIG.01.01.02-00-007/08 in frame of the Polish vaccine Consortium (PVC).

AFIF BEN SALAH got Doctorate in the year 1985 from Faculty of Medicine of Sousse (Tunisia). He worked as Assistant Professor in the field of Medicine/Preventive Medicine, worked as Associate Professor in the field of Epidemiology and also completed his PhD in the year 2000 from Oxford University/ United Kingdom. He is the full Professor of Medicine/Epidemiology, Head of the Laboratory of Epidemiology, Pasteur Institute of Tunis He has published more than 25 papers in reputed journals.

Introduction
The Influenza is an acute viral infection transmitted by air. It`s a highly contagious disease that can cause serious complications, especially among vulnerable people, it presents a major public health issue with a considerable socio-economic impact.
Purpose
- To review the epidemiological situation of influenza in Tunisia in The 2013-214 season.
- Evaluate the experience of Tunisia in the implementation of influenza sentinel surveillance system.
- Make recommendations to overcome challenges.
Methods
This retrospective study is based on data from National surveillance unit of influenza; it relies on descriptive analysis of influenza surveillance data provided by the network of sentinel sites and national influenza centre (NIC).
Results and discussion
Influenza surveillance in Tunisia has been in place since 1996 with the creation of network sentinel sites, but it has developed significantly on March 2014, with the implementation of electronic surveillance system. In Tunisia, clinical, epidemiological and virological surveillance of influenza began in week 40/2013 (1 October 2013) and ended in week 18/2014 (30 April 2014). During the period of study: 156 493 cases ILI (Influenza-like illness) were collected from a total of 2 196 715 patients seen at sentinel sites, representing 7.1% of total patients. The influenza epidemic was spreading in the winter season 2013/2014 during six weeks from 17 December 2013 (2013 / S51) to 17 March 2014 (2014 / S11), it was the shortest of the five seasonal epidemics followed since 2009. The epidemic peak was observed during the week of 4 to 10 February 2014 (2014 / S06), with an incidence rate of 9.5%. On the other hand, Influenza epidemic of 2013-2014 season was mild in intensity and started earlier than that of the previous year. These findings were also observed in Europe and in USA. Among the consultants for influenza-like illness (ILI), 283 severe cases (0.18%) were hospitalized; significantly lower proportion than the previous season (0.25%). The lethality of severe cases remained comparable to that observed in the previous three seasons (0.44%), these severe cases were mainly infected with A (H3N2) virus. All of the 24 governorates of Tunisia have been affected by the influenza and the incidence is higher in regions that are the most populated. Children 5 to 16 years are most affected. Epidemiological surveillance of influenza on the Tunisian territory has shown that the epidemic peak was recorded during the coldest weeks (S6), in fact there is an inverse trend of monthly average temperature and influenza incidence, this remarkable correlation shows that the rate of influenza incidence increased as the temperatures gradually decline. The hypothesis that the virus is favored by low temperatures and inhibited by higher temperatures is confirmed in our study. Influenza virus A (H3N2), A (H1N1) and type B circulated with an initial dominance of type A virus that was reversed in favor of the type B in the last of January. The seasonal distribution of three types of influenza viruses was: Virus A (H3N2) (11.7%), A (H1N1) (0.2%), Virus B (0.2%) and other viruses A non-typed (14.6%).The positivity rate for influenza was 12.1%.
Conclusion
The influenza epidemics 2013-2014 was particularly short, marked by the co-circulation of three influenza viruses, the impact of the epidemic in terms of ILI consultation and hospitalization was less important. Viruses know no borders, control and fight against the influenza requires a global vision of the dynamics of the disease in our country, as well as around the Mediterranean in the context of better contain any unusual events.

Zorraga Mokhtar has completed medicine studies at the age of 28 years from El Manar University, Faculty of Medicine of Tunis. He has Doctoral thesis in Medicine on 5-07-2001, Master of Advanced Studies in Emergency in the Faculty of Medicine of Tunis in 2012, Master of Advanced Studies in Travel medicine in the Faculty of Medicine of Tunis in 2014, ALS (Advanced life support) certificate of European Resuscitation Council in March 2013. He is the coordinator of influenza program in Tunisia, in cooperation with US / CDC, part of InPRIS project (Building and Strengthening Core Capacities for Influenza Preparedness and Response). He has published some papers in reputed journals, the last one in EID journal ( emerging infectious disease) / CDC : “Family Cluster of Middle East Respiratory Syndrome Corona -virus Infections, Tunisia, 2013” on September 2014.

Introduction
The Influenza is an acute viral infection transmitted by air. It`s a highly contagious disease that can cause serious complications, especially among vulnerable people, it presents a major public health issue with a considerable socio-economic impact.
Purpose
- To review the epidemiological situation of influenza in Tunisia in The 2013-214 season.
- Evaluate the experience of Tunisia in the implementation of influenza sentinel surveillance system.
- Make recommendations to overcome challenges.
Methods
This retrospective study is based on data from National surveillance unit of influenza; it relies on descriptive analysis of influenza surveillance data provided by the network of sentinel sites and national influenza centre (NIC).
Results and discussion
Influenza surveillance in Tunisia has been in place since 1996 with the creation of network sentinel sites, but it has developed significantly on March 2014, with the implementation of electronic surveillance system. In Tunisia, clinical, epidemiological and virological surveillance of influenza began in week 40/2013 (1 October 2013) and ended in week 18/2014 (30 April 2014). During the period of study: 156 493 cases ILI (Influenza-like illness) were collected from a total of 2 196 715 patients seen at sentinel sites, representing 7.1% of total patients. The influenza epidemic was spreading in the winter season 2013/2014 during six weeks from 17 December 2013 (2013 / S51) to 17 March 2014 (2014 / S11), it was the shortest of the five seasonal epidemics followed since 2009. The epidemic peak was observed during the week of 4 to 10 February 2014 (2014 / S06), with an incidence rate of 9.5%. On the other hand, Influenza epidemic of 2013-2014 season was mild in intensity and started earlier than that of the previous year. These findings were also observed in Europe and in USA. Among the consultants for influenza-like illness (ILI), 283 severe cases (0.18%) were hospitalized; significantly lower proportion than the previous season (0.25%). The lethality of severe cases remained comparable to that observed in the previous three seasons (0.44%), these severe cases were mainly infected with A (H3N2) virus. All of the 24 governorates of Tunisia have been affected by the influenza and the incidence is higher in regions that are the most populated. Children 5 to 16 years are most affected. Epidemiological surveillance of influenza on the Tunisian territory has shown that the epidemic peak was recorded during the coldest weeks (S6), in fact there is an inverse trend of monthly average temperature and influenza incidence, this remarkable correlation shows that the rate of influenza incidence increased as the temperatures gradually decline. The hypothesis that the virus is favored by low temperatures and inhibited by higher temperatures is confirmed in our study. Influenza virus A (H3N2), A (H1N1) and type B circulated with an initial dominance of type A virus that was reversed in favor of the type B in the last of January. The seasonal distribution of three types of influenza viruses was: Virus A (H3N2) (11.7%), A (H1N1) (0.2%), Virus B (0.2%) and other viruses A non-typed (14.6%).The positivity rate for influenza was 12.1%.
Conclusion
The influenza epidemics 2013-2014 was particularly short, marked by the co-circulation of three influenza viruses, the impact of the epidemic in terms of ILI consultation and hospitalization was less important. Viruses know no borders, control and fight against the influenza requires a global vision of the dynamics of the disease in our country, as well as around the Mediterranean in the context of better contain any unusual events.

Milan Kožíšek is a senior scientist at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague in the group of Jan Konvalinka, Ph.D. He finished there his Ph.D. in 2010 after defense of the thesis “Overcoming drug resistence: The discovery, design and characterization of new nonpeptidic inhibitors of HIV-1 protease“. He is author of 28 papers in peer-reviewed international journals and 4 patents.

The influenza virus causes a severe infection that is responsible for up to half a million deaths each year. The only class of drugs that have been successful targets the viral glycoprotein neuraminidase. Two inhibitors have been approved (Tamiflu, Relenza) and two others have been authorized in various countries for the emergency treatment during pandemics. However, the ongoing development of resistance towards approved inhibitors together with efficient transmission of resistant viruses among humans represent a serious threat to public health.
The approved influenza neuraminidase inhibitors are designed to have (oxa)cyclohexene scaffolds to mimic the oxonium transition state during enzymatic cleavage of sialic acid. Their active forms contain the carboxylate that interacts with three arginine residues in the active site of neuraminidase. Recently, the phosphonate group was successfully used as an isostere of carboxylate in oseltamivir and this compound, tamiphosphor, was discovered as a highly active neuraminidase inhibitor (see review Shie et al., J. Chin. Chem. Soc., 2014). However, the structure of the complex of this promising inhibitor with neuraminidase has not been solved yet and therefore the binding mode into the active site and comparison with oseltamivir carboxylate binding were proposed by molecular docking experiments.
We overexpressed the ectodomain of pandemic neuraminidase (residues 82 to 469) from the influenza virus A/California/07/2009 (H1N1) in Drosophila Schneider S2 cells. The overexpressed neuraminidase with N-terminal tag secreted to the medium was subsequently purified by one-step purification using a streptavidin derivative. The enzyme was first characterized enzymologically by fluorimetric assay using 2´-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MUNANA) as a substrate and the inhibition constants for oseltamivir carboxylate and tamiphosphor were determined, respectively. The binding of the oseltamivir carboxylate or tamiphosphor to the catalytic domain of neuraminidase was thermodynamically characterized by protein microcalorimetry. To our knowledge, this is the first calorimetric determination of the binding mode of an approved neuraminidase inhibitor.Finally, the complex of the neuraminidase ectodomain with tamiphosphor was successfully crystallized, its structure was determined at 1.72 Å resolution and compared with previously reported oseltamivir carboxylate – neuraminidase complex (Vavricka et al., PLoS Pathogens, 2011).
In conclusion, we analyzed enzymologically, thermodynamically and structurally tamiphospor binding to an actual pandemic neuraminidase of H1N1 influenza. The biochemical characterization of tamiphosphor as promising neuraminidase inhibitor could help in structure-based design of more potent neuraminidase inhibitors active against major resistant strains.

Noor Haliza Hasan is a PhD student in Veterinary Sciences at the School of Animal & Veterinary Sciences, The University of Adelaide, Australia. She is currently working on the development of recombinant phage displayed antibodies and antigenic mapping of the M2e protein of avian influenza virus. Attached to the University Malaysia Sabah, Malaysia, she is planning to focus her future research on looking into zoonotic diseases and animal reservoir.

As a part of highly pathogenic avian influenza virus (HPAIV) surveillance strategy, extracellular domain of M2 (M2e) protein is currently explored as positive antigen marker in differentiating antibodies of infected chickens from those which have been vaccinated (DIVA). This study opted to identify the potential of four monoclonal antibodies (mAb) generated through hybridoma technique against the H5N1 M2e peptide for the development of an M2e-based competitive ELISA (M2e c-ELISA). All four mAbs were tested against the sera obtained from chickens experimentally exposed to H5N1 and which were positive for M2e antibodies. Results showed that M2e antibody positive chicken sera compete effectively (90% - 93%) against all three mAbs for binding to the M2e protein. Development of a c-ELISA based on these mAbs could be useful as an alternative diagnostic test for DIVA application in H5N1 surveillance.

Peihua Cao is a PhD candidate in School of Public Health, The University of Hong Kong (2011-present). He has a B.S. in Biostatistics from Southern Medical University in China and was awarded the Outstanding Graduate Student in 2011. His research interests include evolution and epidemiology of influenza viruses, transmission dynamics of infectious diseases, and situation awareness of disease activity using multiple surveillance data sources. He is now working on a multidisciplinary project to integrate influenza epidemiology and genetic data in a subtropical city Hong Kong.

Background
Influenza is an infectious disease that causes significant morbidity and mortality and shows distinct seasonal patterns under different climates. In subtropical and tropical regions such as Hong Kong, influenza shows less clear seasonal patterns, with multiple peaks observed throughout the year. Antigenic variation of influenza viruses has been proposed to play an important role in regulating influenza seasonality.
Methods
We conducted a comprehensive study to understand the characterization of the antigenicity and explore the temporal trend of antigenic changes of influenza A virus (both A/H1N1 and A/H3N2 subtypes) in Hong Kong from January 2013 to December 2014. Clinical samples from 1,013 patients with confirmed A/H1N1 and A/H3N2 influenza viruses were collected from two public hospitals which cover over 80% of total hospitalizations in Hong Kong Island during the study period.
Results
379 specimens were randomly selected for a hemagglutinin amino acids (HA) sequence analysis. Based on sequence analysis, all influenza viruses for both A/H1N1 and A/H3N2 subtypes were characterized by at least 4 fixed amino acid mutations for A/H1N1: P100S, S220T, R240Q, and I338V, and at least 8 fixed amino acid mutations for A/H3N2: H9Y, Q49R, N161S, Q172H, V202G, Y235S, and N294K, with respect to WHO recommended vaccine composition strains A/California/07/2009(for A/H1N1) and A/Victoria/361/2011 (for A/H3N2) in 2013-2014. We further randomly selected 128 specimens (56 A/H1N1 and 72 A/H3N2) for hemagglutinin-inhibition (HI) test. Almost all the HI titer results for both A/H1N1 and A/H3N2 were over 320, which indicated that there were no significant differences between our samples and the reference WHO strains.
Conclusion
The substitutions observed in sequence analysis suggested that there were genetic drifts of the influenza A (both A/H1N1 and A/H3N2 subtypes) viruses from January 2013 to December 2014. Our study may contribute to develop the knowledge of the evolution of influenza A virus circulated in Hong Kong.