MDCK-Adaptive Mutation of A169S Changes Glycosylation Pattern of Hemagglutinin and Enhances MDCK-Based H7N9 Vaccine Virus Production without Loss of Antigenicity and Immunogenicity.

The adaptation of egg-derived H7N9 candidate vaccine virus (CVV) in the mammalian cell line is an approach to developing a high-growth virus strain for the mass production of vaccine manufacturing. The adaptive mutations that occur in hemagglutinin (HA) are critical to the activity and potency of the vaccine virus. Previously, we identified a new mutation of A169S in the HA protein of an MDCK-adapted H7N9 vaccine virus (A/Anhui/2013, RG268); however, whether and how this mutation affects vaccine potency remain to be investigated. In this study, we serially passaged RG268 in MDCK cells and found that the HA titer and the TCID50 of the passaged virus RG268-M5 were 4-fold (HA units/50 µL) and 3.5-fold (log10 TCID50/mL) higher than those of the original CVV. By inspecting tandem MS spectra, we identified a new glycosylation site at N167 near the receptor binding site of the HA protein of RG268-M5. Flow cytometry results revealed that RG268-M5 could efficiently infect MDCK cells and initiate viral protein replication as well as that of RG268. Though the new glycosylation site is in the antigenic epitope of viral HA protein, the HI assay result indicated that the antigenicity of RG268-M5 was similar to RG268. Additionally, immunizing mice with RG268-M5 mixed aluminum hydroxide could induce potent antibody responses against the homologous and heterologous H7N9 viruses in vitro whereas the titers were comparable with those from the RG268 group. These results provide in-depth structural information regarding the effects of site-specific glycosylation on virus properties, which have implications for novel avian influenza vaccine development.

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

IMPORTANCE: By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Characterization and Immunogenicity of Influenza H7N9 Vaccine Antigens Produced Using a Serum-Free Suspension MDCK Cell-Based Platform.

Human infections with avian-origin H7N9 influenza A viruses were first reported in China, and an approximately 38% human mortality rate was described across six waves from February 2013 to September 2018. Vaccination is one of the most cost-effective ways to reduce morbidity and mortality during influenza epidemics and pandemics. Egg-based platforms for the production of influenza vaccines are labor-intensive and unable to meet the surging demand during pandemics. Therefore, cell culture-based technology is becoming the alternative strategy for producing influenza vaccines. The current influenza H7N9 vaccine virus (NIBRG-268), a reassortant virus from A/Anhui/1/2013 (H7N9) and egg-adapted A/PR/8/34 (H1N1) viruses, could grow efficiently in embryonated eggs but not mammalian cells. Moreover, a freezing-dry formulation of influenza H7N9 vaccines with long-term stability will be desirable for pandemic preparedness, as the occurrence of influenza H7N9 pandemics is not predictable. In this study, we adapted a serum-free anchorage-independent suspension Madin-Darby Canine Kidney (MDCK) cell line for producing influenza H7N9 vaccines and compared the biochemical characteristics and immunogenicity of three influenza H7N9 vaccine antigens produced using the suspension MDCK cell-based platform without freeze-drying (S-WO-H7N9), the suspension MDCK cell-based platform with freeze-drying (S-W-H7N9) or the egg-based platform with freeze-drying (E-W-H7N9). We demonstrated these three vaccine antigens have comparable biochemical characteristics. In addition, these three vaccine antigens induced robust and comparable neutralizing antibody (NT; geometric mean between 1016 and 4064) and hemagglutinin-inhibition antibody (HI; geometric mean between 640 and 1613) titers in mice. In conclusion, the serum-free suspension MDCK cell-derived freeze-dried influenza H7N9 vaccine is highly immunogenic in mice, and clinical development is warranted.

Development and Evaluation of Vero Cell-Derived Master Donor Viruses for Influenza Pandemic Preparedness.

The embryonated egg-based platform currently produces the majority of seasonal influenza vaccines by employing a well-developed master donor virus (MDV, A/PR/8/34 (PR8)) to generate high-growth reassortants (HGRs) for A/H1N1 and A/H3N2 subtypes. Although the egg-based platform can supply enough seasonal influenza vaccines, it cannot meet surging demands during influenza pandemics. Therefore, multi-purpose platforms are desirable for pandemic preparedness. The Vero cell-based production platform is widely used for human vaccines and could be a potential multi-purpose platform for pandemic influenza vaccines. However, many wild-type and egg-derived influenza viruses cannot grow efficiently in Vero cells. Therefore, it is critical to develop Vero cell-derived high-growth MDVs for pandemic preparedness. In this study, we evaluated two in-house MDVs (Vero-15 and VB5) and two external MDVs (PR8 and PR8-HY) to generate Vero cell-derived HGRs for five avian influenza viruses (AIVs) with pandemic potentials (H5N1 clade 2.3.4, H5N1 clade 2.3.2.1, American-lineage H5N2, H7N9 first wave and H7N9 fifth wave). Overall, no single MDV could generate HGRs for all five AIVs, but this goal could be achieved by employing two in-house MDVs (vB5 and Vero-15). In immunization studies, mice received two doses of Vero cell-derived inactivated H5N1 and H7N9 whole virus antigens adjuvanted with alum and developed robust antibody responses.

Development of high-growth influenza H7N9 prepandemic candidate vaccine viruses in suspension MDCK cells.

BACKGROUND: Influenza vaccine manufacturers traditionally use egg-derived candidate vaccine viruses (CVVs) to produce high-yield influenza viruses for seasonal or pandemic vaccines; however, these egg-derived CVVs need an adaptation process for the virus to grow in mammalian cells. The low yields of cell-based manufacturing systems using egg-derived CVVs remain an unsolved issue. This study aimed to develop high-growth cell-derived CVVs for MDCK cell-based vaccine manufacturing platforms. METHODS: Four H7N9 CVVs were generated in characterized Vero and adherent MDCK (aMDCK) cells. Furthermore, reassortant viruses were amplified in adherent MDCK (aMDCK) cells with certification, and their growth characteristics were detected in aMDCK cells and new suspension MDCK (sMDCK) cells. Finally, the plaque-forming ability, biosafety, and immunogenicity of H7N9 reassortant viruses were evaluated. RESULTS: The HA titers of these CVVs produced in proprietary suspension MDCK (sMDCK) cells and chicken embryos were 2- to 8-fold higher than those in aMDCK cells. All H7N9 CVVs showed attenuated characteristics by trypsin-dependent plaque assay and chicken embryo lethality test. The alum-adjuvanted NHRI-RG5 (derived from the fifth wave H7N9 virus A/Guangdong/SP440/2017) vaccine had the highest immunogenicity and cross-reactivity among the four H7N9 CVVs. Finally, we found that AddaVax adjuvant improved the cross-reactivity of low pathogenic H7N9 virus against highly pathogenic H7N9 viruses. CONCLUSIONS: Our study indicates that cell-derived H7N9 CVVs possessed high growth rate in new sMDCK cells and low pathogenicity in chicken embryo, and that CVVs generated by this platform are also suitable for both cell- and egg-based prepandemic vaccine production.

Development and characterization of standard reagents for cell-based prepandemic influenza vaccine products.

Outbreaks of infection by novel avian influenza virus strains in humans cause public health issues worldwide, and the development of vaccines against such novel strains is the most effective method for the prevention of these virus outbreaks. All types of vaccines must be tested for potency before use; thus, quantitative potency assays are needed for influenza vaccines. The single radial immunodiffusion (SRID) assay is considered the gold standard for quantification of influenza virus antigens, and the SRID reference reagents are essential for the determination of vaccine potency. However, it remains debatable whether reference reagents derived from egg-based vaccine platforms can be used to precisely quantify non-egg-derived vaccines; thus, influenza vaccine production using cell-based platforms has attracted increasing attention. To evaluate the utility of reference reagents derived from a cell-based influenza vaccine platform, we prepared cell-based reference reagents from MDCK cell-grown viruses and compared them with egg-derived reference reagents. A primary liquid standard (PLS) was purified from cell-derived candidate influenza vaccine viruses, and hemagglutinin (HA) antigen content was determined by a densitometric method. The produced PLS could be stored at 4°C for more than 10 months. We also established a simple HA protein purification method for goat antiserum preparation, and the performance of the resulting antiserum was compared to that of standard reagents obtained using different production platforms. The results of this study indicate that these reference reagents can be used for both cell-based and egg-based production platforms and that the differences between these two types of platforms are negligible.

Seroprevalence of EV-A71 neutralizing antibodies following the 2011 epidemic in HCMC, Vietnam.

Enterovirus-A71 (EV-A71) cyclically causes hand-foot-mouth disease (HFMD) epidemics in Asian children. An EV-A71 epidemic occurred in Southern Vietnam in 2011, but its scale is not clear. We collected residual sera from non-HFMD Vietnamese inpatients in 2012-2013 to determine seroprevalence of EV-A71 neutralizing antibodies, and measured cross-reactive neutralizing antibody titers against three EV-A71 genogroups. About 23.5% of 1-year-old children in Southern Vietnam has been infected by EV-A71, and the median age of infection was estimated to be 3 years. No significant antigenic variation could be detected among the three EV-A71 genogroups. The high seroprevalence of EV-A71 neutralizing antibody in children living in southern Vietnam indicates the necessity of introducing EV-A71 vaccines in southern Vietnam, particularly for children under 6 months of age. Moreover, it is critical to understand EV-A71 disease burden for formulating national vaccination policy.

Establishment of Asia-Pacific Network for Enterovirus Surveillance.

Enteroviruses (EV), the major pathogens of hand, foot, and mouth disease (HFMD) and herpangina, affect millions of children each year. Most human enteroviruses cause self-limited infections except polioviruses, enterovirus A71 (EV-A71), enterovirus D68 (EV-D68), and several echoviruses (Echo) and coxsackieviruses (CV). Especially, EV-A71 has repeatedly caused large-scale outbreaks in the Asia-Pacific region since 1997. Some Asian countries have experienced cyclical outbreaks of severe EV-A71 infections and initiated development of EV-A71 vaccines. Five EV-A71 vaccine candidates have been clinically evaluated and three of them were approved for marketing in China. However, none of the China-approved products seek marketing approval in other countries. This situation supports a role for collaboration among Asian countries to facilitate clinical trials and licensure of EV-A71 vaccines. Additionally, enterovirus D68 outbreaks have been reported in the US and Taiwan currently and caused severe complications and deaths. Hence, an Asia-Pacific Network for Enterovirus Surveillance (APNES) has been established to estimate disease burden, understand virus evolution, and facilitate vaccine development through harmonizing laboratory diagnosis and data collection. Founded in 2017, the APNES is comprised of internationally recognized experts in the field of enterovirus in Asian countries working to raise awareness of this potentially fatal and debilitating disease. This article demonstrated the summaries of the first expert meeting, 2017 International Workshop on Enterovirus Surveillance and Vaccine Development, held by APNES in Taipei, Taiwan, March 2017.

Process development for pandemic influenza VLP vaccine production using a baculovirus expression system.

BACKGROUND: Influenza viruses cause hundreds of thousands of respiratory diseases worldwide each year, and vaccination is considered the most effective approach for preventing influenza annual epidemics or pandemics. Since 1950, chicken embryonated eggs have been used as the main method for producing seasonal influenza vaccines. However, this platform has the main drawback of a lack of scale-up flexibility, and thus, egg-based vaccine manufacturers cannot supply sufficient doses within a short period for use for pandemic prevention. As a result, strategies for reducing the manufacturing time and increasing production capacity are urgently needed. Non-virion vaccine methods have been considered an alternative strategy against an influenza pandemic, and the purpose of maintaining an immunogenic capsule structure with infectious properties appears to be met by the virus-like particle (VLP) platform. RESULTS: An influenza H7N9-TW VLP production platform using insect cells, which included the expression of hemagglutinin (HA), NA, and M1 proteins, was established. To scale up H7N9-TW VLP production, several culture conditions were optimized to obtain a higher production yield. A high level of dissolved oxygen (DO) could be critical to H7N9-TW VLP production. If the DO was maintained at a high level, the HA titer obtained in the spinner flask system with ventilation was similar to that obtained in a shake flask. In this study, the HA titer in a 5-L bioreactor with a well-controlled DO level was substantially improved by 128-fold (from 4 HA units (HAU)/50 µL to 512 HAU/50 µL). CONCLUSIONS: In this study, a multigene expression platform and an effective upstream process were developed. Notably, a high H7N9-TW VLP yield was achieved using a two-step production strategy while a high DO level was maintained. The upstream process, which resulted in high VLP titers, could be further used for large-scale influenza VLP vaccine production.

Evaluation of novel disposable bioreactors on pandemic influenza virus production.

Since 1997, the highly pathogenic influenza H5N1 virus has spread from Hong Kong. According to the WHO bulletin report, the H5N1 virus is a zoonotic disease threat that has infected more than 850 humans, causing over 450 deaths. In addition, an outbreak of another new and highly pathogenic influenza virus (H7N9) occurred in 2013 in China. These highly pathogenic influenza viruses could potentially cause a worldwide pandemic. it is crucial to develop a rapid production platform to meet this surge demand against any possible influenza pandemic. A potential solution for this problem is the use of cell-based bioreactors for rapid vaccine production. These novel bioreactors, used for cell-based vaccine production, possess various advantages. For example, they enable a short production time, allow for the handling highly pathogenic influenza in closed environments, and can be easily scaled up. In this study, two novel disposable cell-based bioreactors, BelloCell and TideCell, were used to produce H5N1 clade II and H7N9 candidate vaccine viruses (CVVs). Madin-Darby canine kidney (MDCK) cells were used for the production of these influenza CVVs. A novel bench-scale bioreactor named BelloCell bioreactor was used in the study. All culturing conditions were tested and scaled to 10 L industrial-scale bioreactor known as TideCell002. The performances of between BelloCell and TideCell were similar in cell growth, the average MDCK cell doubling time was slightly decreased to 25 hours. The systems yielded approximately 39.2 and 18.0 µg/ml of HA protein with the 10-liter TideCell002 from the H5N1 clade II and H7N9 CVVs, respectively. The results of this study not only highlight the overall effectiveness of these bioreactors but also illustrate the potential of maintaining the same outcome when scaled up to industrial production, which has many implications for faster vaccine production. Although additional studies are required for process optimization, the results of this study are promising and show that oscillating bioreactors may be a suitable platform for pandemic influenza virus production.

The stability and immunogenicity of inactivated MDCK cell-derived influenza H7N9 viruses.

In recent years, cell-based influenza vaccines have gained a great interest over the egg-based vaccines. Several inactivated H7N9 vaccines have been evaluated in clinical trials, including whole-virion vaccines, split vaccines and subunit vaccines. Recently, we developed a new suspension MDCK (sMDCK) cell line for influenza viruses production. However, the properties of purified antigen from sMDCK cells remain unclear. In this study, the stability of influenza H7N9 vaccine bulk derived from sMDCK cells was investigated, and the data were compared with the vaccine antigen derived from our characterized adhesion MDCK (aMDCK) cells in serum-free medium. The influenza H7N9 bulks derived from sMDCK and aMDCK cells were stored at 2-8 °C for different periods of time, and a number of parameters selected to monitor the H7N9 vaccine antigen stability were evaluated at each interval (1, 3 and 12 months). The monitored parameters included virus morphology, hemagglutinin (HA) activity, HA concentration, antigenicity, and immunogenicity. The sMDCK-derived H7N9 bulk showed similar morphology to that of the aMDCK-derived H7N9 bulk, and there were no obvious changes after the extended storage periods. Furthermore, the HA titer, HA concentration, and antigenicity of sMDCK-derived H7N9 bulk were stable after 28 months of storage. Finally, the results of hemagglutination inhibition and neutralization tests showed that sMDCK- and aMDCK-derived H7N9 vaccines had comparable immunogenicity. These results indicated that sMDCK-derived H7N9 bulk has good stability compared to that of aMDCK-derived H7N9 bulk. Thus, the newly developed suspension MDCK cell line shows a great alternative for manufacturing cell-based influenza vaccines.

Genomic analysis of serologically untypable human enteroviruses in Taiwan.

BACKGROUND: Human enteroviruses contain over 100 serotypes. We have routinely conducted enterovirus surveillance in northern Taiwan; but about 10% of isolates could not be serotyped using traditional assays. Next-generation sequencing (NGS) is a powerful tool for genome sequencing. METHODS: In this study, we established an NGS platform to conduct genome sequencing for the serologically untypable enterovirus isolates. RESULTS: Among 130 serologically untypable isolates, 121 (93%) of them were classified into 29 serotypes using CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer)-based RT-PCR to amplify VP1 genes (VP1-CODEHOP). We further selected 52 samples for NGS and identified 59 genome sequences from 51 samples, including 8 samples containing two virus genomes. We also detected 23 genome variants (nucleotide identity < 90% compared with genome sequences in the public domain) which were potential genetic recombination, including 9 inter-serotype recombinants and 14 strains with unknown sources of recombination. CONCLUSIONS: We successfully integrated VP1-CODEHOP and NGS techniques to conduct genomic analysis of serologically untypable enteroviruses.

Development of American-Lineage Influenza H5N2 Reassortant Vaccine Viruses for Pandemic Preparedness.

Novel low-pathogenic avian influenza (LPAI) H5N2 viruses hit poultry farms in Taiwan in 2003, and evolved into highly pathogenic avian influenza (HPAI) viruses in 2010. These viruses are reassortant viruses containing HA and NA genes from American-lineage H5N2 and six internal genes from local H6N1 viruses. According to a serological survey, the Taiwan H5N2 viruses can cause asymptomatic infections in poultry workers. Therefore, a development of influenza H5N2 vaccines is desirable for pandemic preparation. In this study, we employed reverse genetics to generate a vaccine virus having HA and NA genes from A/Chicken/CY/A2628/2012 (E7, LPAI) and six internal genes from a Vero cell-adapted high-growth H5N1 vaccine virus (Vero-15). The reassortant H5N2 vaccine virus, E7-V15, presented high-growth efficiency in Vero cells (512 HAU, 107.6 TCID50/mL), and passed all tests for qualification of candidate vaccine viruses. In ferret immunization, two doses of inactivated whole virus antigens (3 µg of HA protein) adjuvanted with alum could induce robust antibody response (HI titre 113.14). In conclusion, we have established reverse genetics to generate a qualified reassortant H5N2 vaccine virus for further development.

Improving immunogenicity of influenza virus H7N9 recombinant hemagglutinin for vaccine development.

Human infections of novel avian influenza A virus (H7N9) emerged in early 2013 and caused about 40% case-fatality through 2017. Therefore, development of influenza H7N9 vaccines is critical for pandemic preparedness. Currently, there are three means of production of commercial influenza vaccines: egg-based, mammalian cell-based, and insect cell-based platforms. The insect cell-based platform has the advantage of high speed in producing recombinant protein. In this study, we evaluate the stability and immunogenicity of two different influenza H7 HA expression constructs generated using the baculovirus system, including membrane-based full-length HA (mH7) and secreted ectodomain-based H7 (sH7). The mH7 construct could form an oligomer-rosette structure and had a high hemagglutinin (HA) titer 8192. In contrast to mH7, the sH7 construct could not form an oligomer-rosette structure and did not have HA titer before cross-linking with anti-His antibody. Thermal stability tests showed that the sH7 and mH7 constructs were unstable at 43 °C and 52 °C, respectively. In a mice immunization study, the mH7 construct but not the sH7 construct could induce robust HI and neutralizing antibody titers. In conclusion, further development of the mH7 vaccine candidate is desirable.

Application of stable isotope dimethyl labeling for MRM based absolute antigen quantification of influenza vaccine.

Determining the precursor/product ion pair and optimal collision energy are the critical steps for developing a multiple reaction monitoring (MRM) assay using triple quadruple mass spectrometer for protein quantitation. In this study, a platform consisting of stable isotope dimethyl labeling coupled with triple-quadruple mass spectrometer was used to quantify the protein components of the influenza vaccines. Dimethyl labeling of both the peptide N-termini and the ϵ-amino group of lysine residues was achieved by reductive amination using formaldehyde and sodium cyanoborohydrate. Dimethylated peptides are known to exhibit dominant a1 ions under gas phase fragmentation in a mass spectrometer. These a1 ions can be predicted from the peptide N-terminal amino acids, and their signals do not vary significantly across a wide range of collision energies, which facilitates the determination of MRM transition settings for multiple protein targets. The intrinsic a1 ions provide sensitivity for acquiring MRM peaks that is superior to that of the typical b/y ions used for native peptides, and they also provided good linearity (R2 ≥ 0.99) at the detected concentration range for each peptide. These features allow for the simultaneous quantification of hemagglutinin and neuraminidase in vaccines derived from either embryo eggs or cell cultivation. Moreover, the low abundant ovalbumin residue originated from the manufacturing process can also be determined. The results demonstrate that the stable isotope dimethyl labeling coupled with MRM Mass spectrometry screening of a1 ions (i.e., SIDa-MS) can be used as a high-throughput platform for multiple protein quantification of vaccine products.

Genomic analysis of enterovirus D68, including one strain isolated from a child with Wilson's disease in Taiwan.

North America experienced life-threatening outbreaks of enterovirus D68 (EV-D68) in 2014. We retrospectively detected EV-D68 from a child with Wilson's disease in 2008 in Taiwan. After comparing this EV-D68/Taiwan/2008 strain with EV-D68 genomes obtained from the public domain, it was classified as genome type 1-B; it is phylogenetically related to the predominant EV-D68 viruses that circulated in 2009 in Vietnam. It is necessary to strengthen EV-D68 detection globally, including in children with acute liver failure. Moreover, harmonization of genomic analysis of EV-D68 is desirable to understand global evolution of EV-D68.

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses.

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries. Currently, five vaccine candidates have been evaluated in clinical trials in China (three C4 candidates), Singapore (one B2 candidate), and Taiwan (one B4 candidate). Overall, the peak viral titers of these 5 vaccine candidates could only reach about 107 TCID50/mL. Moreover, genotypes of these 5 candidates are different from the current predominant genotype B5 in Taiwan and South-Eastern Asia. We adapted a high-growth EV71 genotype B5 (HG-B5) virus after multiple passages and plaque selections in Vero cells and the HG-B5 virus could reach high titers (>108 TCID50/mL) in a microcarrier-based cell culture system. The viral particles were further purified and formulated with alum adjuvant. After two doses of intramuscular immunization in rabbits, the HG-B5 vaccine candidate could induce cross-reactive neutralizing antibodies against the three major EV71 genogroups. In conclusion, a high-growth EV71 virus was successfully adapted in Vero cells and could induce broad spectrum neutralizing antibody titers against three (A, B5, and C4) genotypes in rabbits.

A fast and efficient purification platform for cell-based influenza viruses by flow-through chromatography.

Since newly emerging influenza viruses with pandemic potentials occurred in recent years, the demand for producing pandemic influenza vaccines for human use is high. For the development of a quick and efficient vaccine production, we proposed an efficient purification platform from the harvest to the purified bulk for the cell-based influenza vaccine production. This platform based on flow-through chromatography and filtration steps and the process only involves a few purification steps, including depth filtration, inactivation by formaldehyde, microfiltration, ultrafiltration, anion-exchange and ligand-core chromatography and sterile filtration. In addition, in the proposed chromatography steps, no virus capture steps were employed, and the purification results were not affected by the virus strain variation, host cells and culturing systems. The results from different virus strains which produced by Vero or MDCK cells in different culturing systems also obtained 33-46% HA recovery yields by this platform. The overall removal rates of the protein and DNA concentration in the purified bulk were over 96.1% and 99.7%, respectively. The low residual cellular DNA concentrations were obtained ranged from 30 to 130pg per human dose (15µg/dose). All influenza H5N1 purified bulks met the regulatory requirements for human vaccine use.

A mutation-resistant deoxyribozyme OR gate for highly selective detection of viral nucleic acids.

Highly selective probes hybridize only to fully complementary DNA or RNA sequences and, therefore, often fail to recognize mutated viral genomes. Here we designed a probe that possesses two seemingly incompatible properties: it tolerates some point mutations in genome, while it remains selective towards others. An OR deoxyribozyme logic gate was designed to fluorescently report the sequences of enterovirus 71 (EV71) covering ∼90% of all known EV71 strains. Importantly, sequences of closely related coxsackieviruses that differed by single nucleotides were reliably differentiated in 7 out of 8 cases.

Assessment of pathogenicity and antigenicity of American lineage influenza H5N2 viruses in Taiwan.

During December 2003 and March 2004, large scale epidemics of low-pathogenic avian influenza (LPAI) H5N2 occurred in poultry farms in central and southern Taiwan. Based on genomic analysis, these H5N2 viruses contain HA and NA genes of American-lineage H5N2 viruses and six internal genes from avian influenza A/H6N1 viruses endemic in poultry in Taiwan. After disappearing for several years, these novel influenza H5N2 viruses caused outbreaks in poultry farms again in 2008, 2010 and 2012, and have evolved into high pathogenic AI (HPAI) since 2010. Moreover, asymptomatic infections of influenza H5N2 were detected serologically in poultry workers in 2012. Therefore, we evaluated antigenicity and pathogenicity of the novel H5N2 viruses in ferrets. We found that no significant antigenic difference was detected among the novel H5N2 viruses isolated from 2003 to 2014 and the novel H5N2 viruses could cause mild infections in ferrets. Monitoring zoonotic transmission of the novel H5N2 viruses is necessary.