Avian H5 Research Articles

An SYBR Green-based real-time RT-PCR assay for the detection of H5 hemagglutinin subtype avian influenza virus

Increasing diversity among H5 hemagglutinin (HA) subtype avian influenza (AI) viruses has resulted in the need of novel sensitive and specific molecular assays. In this study, an SYBR Green-based real-time reverse transcription-PCR (RRT-PCR) assay was developed for the detection of H5 subtype AI virus. Sequence analysis of the Mexican lineage H5N2 isolates (subgroup B) revealed several mismatches in the primer/hydrolysis probe set reported in the commonly used RRT-PCR assay for the detection of H5 North American lineage. The present assay was designed to circumvent the challenge that these viruses represent for the specific detection of H5 subtype AI viruses. This RRT-PCR assay successfully detected a range of different H5 subtype AI strains from both Eurasian and North American lineages representing different avian H5 HA clades from diverse geographical locations. The sensitivity of the present method was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection of 100 50% egg infectious dose (EID50)/mL and 4.2 gene copies/μl. The linear ranges of the assay span within 106–100 EID50/mL and 106–100 gene copies/μl. The results obtained from this method were directly compared with those of the H5 RRT-PCR assay recommended by the OIE. The comparison was performed with 110 tracheal and cloacal swabs from various bird species collected during field and laboratory investigations in Eurasia and Africa in 2006 and 2008 and showed 100% agreement. This assay is recommended as an alternative method, also allowing a ‘double check’ approach detection, to be use mainly in outbreak scenarios with higher risk of poultry infections by Central American/Caribbean H5 AI viruses.

Detection of Avian Influenza Viruses and Differentiation of H5, H7, N1, and N2 Subtypes Using a Multiplex Microsphere Assay

In an outbreak of highly pathogenic H5 and H7 avian influenza, rapid analysis of a large number of clinical samples with the potential to rapidly identify the virus subtype is extremely important. Herein, we report on the development of a rapid multiplex microsphere assay for the simultaneous detection of all avian influenza viruses (AIV) as well as the differentiation of H5, H7, N1, and N2 subtypes. A reverse transcriptase-PCR (RT-PCR) reaction, followed by hybridization of the amplified product with specific oligonucleotide probe-coated microspheres, was conducted in a multiplex format. Following incubation with a reporter dye, the fluorescence intensity was measured using a suspension array system. The limit of detection of the probe-coupled microspheres ranged from 1 x 10(5) to 1 x 10(9) copies of RT-PCR amplified product and the sensitivity of the multiplex assay ranged from 1 x 10(2.5) to 1 x 10(3.2) 50% embryo infectious doses of virus. The diagnostic accuracy of the assay, compared to the standard real-time RT-PCR, was evaluated using 102 swab samples from chickens exposed to low pathogenic AIV, and 97.05% of samples gave identical results with both the assays. The calculated specificity of the assay was 97.43%. Although the assay still needs to be validated, it appears to be a suitable diagnostic tool for detection and differentiation of avian influenza virus H5, H7, N1, and N2 subtypes.

A multiplex RT-PCR assay for detection and differentiation of avian H3, H5, and H9 subtype influenza viruses and Newcastle disease viruses

Avian influenza viruses (AIVs) and Newcastle disease viruses (NDVs) co-circulate in the poultry population in China. These viruses cause repeated disease outbreaks that exhibit similar clinical symptoms and epidemiological patterns. H5 and H9 influenza viruses are the major pathogens infecting poultry stocks. Recently, H3 AIV (one of the main subtypes in waterfowl) has become endemic in chickens. A multiplex reverse-transcriptase polymerase chain reaction (mRT-PCR) assay was designed for simultaneous detection and differentiation of avian H3, H5, H9 subtype AIVs and NDVs. Four primer sets were evaluated, three of which specifically targeted the hemagglutinin genes of H3, H5 and H9 AIVs, while the other targeted the NDV fusion gene. The sensitivity and specificity of the mRT-PCR assay was determined. The assay detected the major clades or genotypes of all of the reference AIVs and NDVs currently circulating in China. In addition, the mRT-PCR results obtained from screening 380 clinical swabs and 12 experimental tracheal samples were consistent with those obtained using conventional virus isolation methods. The mRT-PCR assay was established successfully for the detection and differentiation of avian H3, H5, and H9 subtype AIVs and NDVs. The method should, therefore, provide a valuable diagnostic tool for these infections.

Receptor-Binding Profiles of H7 Subtype Influenza Viruses in Different Host Species

Influenza viruses of gallinaceous poultry and wild aquatic birds usually have distinguishable receptor-binding properties. Here we used a panel of synthetic sialylglycopolymers and solid-phase receptor-binding assays to characterize receptor-binding profiles of about 70 H7 influenza viruses isolated from aquatic birds, land-based poultry, and horses in Eurasia and America. Unlike typical duck influenza viruses with non-H7 hemagglutinin (HA), all avian H7 influenza viruses, irrespective of the host species, displayed a poultry-virus-like binding specificity, i.e., preferential binding to sulfated oligosaccharides Neu5Acα2-3Galβ1-4(6-O-HSO(3))GlcNAc and Neu5Acα2-3Galβ1-4(Fucα1-3)(6-O-HSO(3))GlcNAc. This phenotype correlated with the unique amino acid sequence of the amino acid 185 to 189 loop of H7 HA and seemed to be dependent on ionic interactions between the sulfate group of the receptor and Lys193 and on the lack of sterical clashes between the fucose residue and Gln222. Many North American and Eurasian H7 influenza viruses displayed weak but detectable binding to the human-type receptor moiety Neu5Acα2-6Galβ1-4GlcNAc, highlighting the potential of H7 influenza viruses for avian-to-human transmission. Equine H7 influenza viruses differed from other viruses by preferential binding to the N-glycolyl form of sialic acid. Our data suggest that the receptor-binding site of contemporary H7 influenza viruses in aquatic and terrestrial birds was formed after the introduction of their common precursor from ducks to a new host, presumably, gallinaceous poultry. The uniformity of the receptor-binding profile of H7 influenza viruses in various wild and domestic birds indicates that there is no strong receptor-mediated host range restriction in birds on viruses with this HA subtype. This notion agrees with repeated interspecies transmission of H7 influenza viruses from aquatic birds to poultry.

Inactivation of high and low pathogenic avian influenza virus H5 subtypes by copper ions incorporated in zeolite-textile materials

The effect of cotton textiles containing Cu2+ held by zeolites (CuZeo-textile) on the inactivation of H5 subtype viruses was examined. Allantoic fluid (AF) containing a virus (AF virus) (0.1ml) was applied to the textile (3×3-cm), and incubated for a specific period at ambient temperature. After each incubation, 0.9ml of culture medium was added followed by squeezing to recover the virus into the medium. The recovered virus was titrated using Madin–Darby canine kidney (MDCK) cells or 10-day-old embryonated chicken eggs.The highly pathogenic H5N1 and the low pathogenic H5N3 viruses were inactivated on the CuZeo-textile, even after short incubation. The titer of A/chicken/Yamaguchi/7/04 (H5N1) in MDCK cells and in eggs declined by >5.0 log10 and 5.0log10, respectively, in 30s. The titer of A/whooper swan/Hokkaido/1/08 (H5N1) in MDCK cells declined by 2.3 and 3.5 in 1 and 5min, respectively. When A/whistling swan/Shimane/499/83 (H5N3) was treated on the CuZeo-textile for 10min, the titer declined by >5.0log10 in MDCK cells and by >3.5log10 in eggs. In contrast, no decrease in the titers was observed on cotton textiles containing zeolites alone (Zeo-textile). Neither cytopathic effects nor NP antigens were detected in MDCK cells inoculated with the H5N1 virus treated on the CuZeo-textile. The viral genes (H5, N1, M, and NP) were amplified from the virus treated on the CuZeo-textile by RT-PCR. The hemagglutinating activity of the CuZeo-textile treated virus was unaffected, indicating that virus–receptor interactions were maintained. Electron microscopic analysis revealed a small number of particles with morphological abnormalities in the H5N3 virus samples recovered immediately from the CuZeo-textile, while no particles were detectable in the 10-min treated sample, suggesting the rapid destruction of virions by the Cu2+ in the CuZeo-textile. The loss of infectivity of H5 viruses could, therefore, be due to the destruction of virions by Cu2+. Interestingly, CuCl2 treatment (500 and 5000μM) did not have an antiviral effect on the AF viruses (H5N1 and H5N3) even after 48h of incubation, although the titer of the purified H5N3 virus treated with CuCl2 declined greatly. The antiviral effect was inhibited by adding the AF to the purified H5N3 virus prior to the CuCl2 treatment. The known antibacterial/antifungal activities of copper suggest that the CuZeo-textile can be applied at a high level of hygiene in both animals and humans.

Simultaneous subtyping and pathotyping of the 2010–2011 South Korean HPAI outbreak strain by using a diagnostic microarray

The highly pathogenic avian influenza (HPAI) subtype H5N1 virus has caused continuous outbreaks in the poultry industry with devastating economic losses and is a severe threat to public health. In the present study, we developed a low-density microarray for the rapid detection and identification of avian influenza virus hemagglutinin subtypes H5, H7, and H9 and neuraminidase subtypes N1, N2, and N3. We evaluated this diagnostic microarray using 6 HPAI H5N1 clade 2.3.2 viruses that caused the South Korean outbreaks during the 2010–2011 winter season. Cy3-labeled DNA targets were generated by reverse transcription polymerase chain reaction (RT-PCR) using Cy3-labeled universal primers. The Cy3-labeled RTPCR products were then hybridized to the microarray. All subtypes and pathotypes were correctly determined and were found to be identical to the nucleotide sequencing results. This diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of avian influenza viruses.

Vaccination with drifted variants of avian H5 hemagglutinin protein elicits a broadened antibody response that is protective against challenge with homologous or drifted live H5 influenza virus

Substantial H5 influenza HA directed immunity is elicited after vaccination of human subjects who had been previously immunized with a drifted H5 HA variant. We sought to investigate the characteristics of H5 HA specific immune responses in more depth by developing an animal model of H5 HA vaccination using drift variants of recombinant H5 HA proteins. HA proteins derived from influenzas A/Vietnam/1203/04 (Clade 1) and A/Indonesia/05/05 (Clade 2.1) were chosen. The sequence of vaccination consisted of two doses of homologous protein, followed by one additional dose of the homologous or heterologous, drifted HA protein. Each dose of HA was combined with CpG as an adjuvant and was injected subcutaneously. All the animals exhibited a serum IgG antibody response that cross-reacted with both HAs in an ELISA. However, those animals that received the drifted variant exhibited higher reactivity to the heterologous HA. Competitive ELISA of serum from drift-variant recipients showed evidence of antibody focusing towards the drifted HA, suggesting modification of the response towards improved cross-reactivity, though development of neutralizing antibodies was limited. Nevertheless, animals were protected against live-virus challenge, and passive transfer of serum was sufficient to confer protection to otherwise naïve mice, indicating that both neutralizing and non-neutralizing antibodies offer some degree of protection. These findings suggest that pre-vaccination against H5 influenza has the potential to prime immunity against emerging drifted H5 strains, and could also lower the dose requirements of vaccination in the event of a pandemic.

Development of a reverse transcription loop-mediated isothermal amplification method for the rapid detection of avian influenza virus subtype H7

A rapid and sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of the H7 avian influenza virus (H7 AIV) isotype was developed. The minimum detection limit of the RT-LAMP assay was 0.1-0.01 PFU per reaction for H7 AIV RNA, making this assay 100-fold more sensitive than the conventional RT-PCR method. This RT-LAMP assay also has the capacity to detect both high- and low-pathogenic H7 AIV strains. Using a pool of RNAs extracted from influenza viruses corresponding to all 15 HA subtypes (in addition to other avian pathogenic viruses), the RT-LAMP system was confirmed to amplify only H7 AIV RNA. Furthermore, specific pathogen free (SPF) chickens were infected artificially with H7 AIV, throat and cloacal swabs were collected, and viral shedding was examined using viral isolation, RT-PCR and RT-LAMP. Shedding was detected following viral isolation and RT-LAMP one day after infection, whereas viral detection using RT-PCR was effective only on day 3 post-infection. These results indicate that the RT-LAMP method could facilitate epidemiological surveillance and the rapid diagnosis of the avian influenza subtype H7.

Rapid PCR-Based Molecular Pathotyping of H5 and H7 Avian Influenza Viruses

While the majority of avian influenza virus (AIV) subtypes are classified as low-pathogenicity avian influenza viruses (LPAIV), the H5 and H7 subtypes have the ability to mutate to highly pathogenic avian influenza viruses (HPAIV) in poultry and therefore are the etiological agents of notifiable AIV (NAIV). It is of great importance to distinguish HPAIV from LPAIV variants during H5/H7 outbreaks and surveillance. To this end, a novel and fast strategy for the molecular pathotyping of H5/H7 AIVs is presented. The differentiation of the characteristic hemagglutinin (HA) protein cleavage sites (CSs) of HPAIVs and LPAIVs is achieved by a novel PCR method where the samples are interrogated for all existing CSs with a 484-plex primer mixture directly targeting the CS region. CSs characteristic for HP or LP H5/H7 viruses are distinguished in a seminested duplex real-time PCR format using plexor fluorogenic primers. Eighty-six laboratory isolates and 60 characterized NAIV-positive clinical specimens from poultry infected with H5/H7 both experimentally and in the field were successfully pathotyped in the validation. The method has the potential to substitute CS sequencing in the HA gene for the determination of the molecular pathotype, thereby providing a rapid means to acquire additional information concerning NAIV outbreaks, which may be critical to their management. The new assay may be extended to the LP/HP differentiation of previously unknown H5/H7 isolates. It may be considered for integration into surveillance and control programs in both domestic and wild bird populations.

Coexpression of Avian Influenza Virus H5 and N1 by Recombinant Newcastle Disease Virus and the Impact on Immune Response in Chickens

Coexpression of Avian Influenza Virus H5 and N1 by Recombinant Newcastle Disease Virus and the Impact on Immune Response in Chickens

Coexpression of Avian Influenza Virus H5 and N1 by Recombinant Newcastle Disease Virus and the Impact on Immune Response in Chickens

To analyze the contribution of neuraminidase (NA) toward protection against avian influenza virus (AIV) infection, three different recombinant Newcastle disease viruses (NDVs) expressing hemagglutinin (HA) or NA, or both, of highly pathogenic avian influenza virus (HPAIV) were generated. The lentogenic NDV Clone 30 was used as backbone for the insertion of HA of HPAIV strain A/chicken/Vietnam/P41/05 (H5N1) and NA of HPAIV strain A/duck/Vietnam/TG24-01/05 (H5N1). The HA was inserted between the genes encoding NDV phosphoprotein (P) and matrixprotein (M), and the NA was inserted between the fusion (F) and hemagglutinin-neuraminidase protein (HN) genes, resulting in NDVH5VmPMN1FHN. Two additional recombinants were constructed carrying the HA gene between the NDV P and M genes (NDVH5VmPM) or the NA between F and HN (NDVN1FHN). All recombinants replicated well and stably expressed the HA gene, the NA gene, or both. Chickens immunized with NDVH5VmPMN1FHN or NDVH5VmPM were protected against two different HPAIV H5N1 and also against HPAIV H5N2. In contrast, immunization of chickens with NDVN1FHN induced NDV- and AIV N1-specific antibodies but did not protect the animals against a lethal dose of HPAIV H5N1. Furthermore, expression of AIV N1, in addition to AIV H5 by NDV, did not increase protection against HPAIV H5N1.

Reverse genetics plasmid for cloning unstable Influenza A virus gene segments

Reverse genetics approaches that enable the generation of recombinant influenza A viruses entirely from plasmids are invaluable for studies on virus replication, morphogenesis, pathogenesis, or transmission. Furthermore, influenza virus reverse genetics is now critical for the development of new vaccines for this human and animal pathogen. Periodically, influenza gene segments are unstable within plasmids in bacteria. The PB2 gene segment of a highly pathogenic avian H5 influenza virus A/Turkey/Ontario/7732/1966 (Ty/Ont) was unstable in commonly available cloning plasmids (e.g., pcDNA3.1/V5-His-TOPO) and in standard influenza virus reverse genetics plasmids (e.g., pHH21), which contain high copy origins of replication. Thus, a low-copy influenza reverse genetics plasmid (pGJ3C3) was developed to enable rapid cloning of unstable influenza A virus genes using ligation-independent recombination-based cloning. The unstable Ty/Ont PB2 gene segment was efficiently cloned using the pGJ3C3 plasmid and this clone was used to rescue a recombinant Ty/Ont virus. This low copy reverse genetics plasmid will be useful for cloning other unstable segments of influenza A viruses in order to rescue recombinant viruses, which will facilitate basic studies and vaccine seed stock production.

Upstream start codon in segment 4 of North American H2 avian influenza A viruses

H2N2 influenza A virus was the cause of the 1957 pandemic. Due to its constant presence in birds, the H2 subtype remains a topic of interest. In this work, comparison of H2 leader sequences of influenza A segment 4 revealed the presence of an upstream in-frame start codon in a majority of North American avian strains. This AUG is located seven codons upstream of the conventional start codon and is in a good Kozak context. In vivo experiments, using a luciferase reporter gene fused to leader sequences derived from North American avian H2 strains, support the efficient use of the upstream start codon. These results were corroborated by in vitro translation data using full-length segment 4 mRNA. Phylogenic analyses indicate that the upstream AUG, first detected in 1976, is stably nested in the North American avian lineage of H2 strains nowadays. The possible consequences of the upstream AUG are discussed.

Adjuvant-Free Immunization with Hemagglutinin-Fc Fusion Proteins as an Approach to Influenza Vaccines

The hemagglutinins (HAs) of human H1 and H3 influenza viruses and avian H5 influenza virus were produced as recombinant fusion proteins with the human immunoglobulin Fc domain. Recombinant HA-human immunoglobulin Fc domain (HA-HuFc) proteins were secreted from baculovirus-infected insect cells as glycosylated oligomer HAs of the anticipated molecular mass, agglutinated red blood cells, were purified on protein A, and were used to immunize mice in the absence of adjuvant. Immunogenicity was demonstrated for all subtypes, with the serum samples demonstrating subtype-specific hemagglutination inhibition, epitope specificity similar to that seen with virus infection, and neutralization. HuFc-tagged HAs are potential candidates for gene-to-vaccine approaches to influenza vaccination.

Surveillance for avian influenza virus subtypes H5 and H7 in chickens and turkeys farmed commercially in New Zealand

AIM: To determine the status of avian influenza (AI) virus subtypes H5 and H7 of New Zealand's commercial chicken and turkey farms. METHODS: A cross-sectional serological survey, stratified by production sector, used a sample frame defined by those farms registered with the Poultry Industry Association of New Zealand (PIANZ) or the Egg Producers Federation of New Zealand (EPF). Sectors included were chicken broiler, caged/barn layer, free-range layer, pullet rearer and turkey broiler. The survey used a between- and within-farm design prevalence of 5% (95% confidence for chickens, 99% confidence for turkeys) and 30% (95% confidence), respectively, of AI virus subtypes H5 and H7. The epidemiological unit was the farm for the free-range layer sector, and the individual shed/barn for the other sectors. Serum samples were screened using a commercial generic influenza A indirect ELISA; positive samples were subjected to haemagglutination-inhibition (HI) testing for AI virus subtypes H5 and H7. A comprehensive investigation, that included widespread serological and antigenic screening, was carried out on all farms identified with serum reactors to either the H5 or H7 virus subtype. RESULTS: A total of 4,180 blood samples from 167 chicken and 10 turkey farms were collected and tested using ELISA. Positive ELISA results were returned from 26 farms, comprising 10 caged/barn layer, 14 free-range layer and two turkey (shed-raised) broiler farms. HI testing of ELISA-positive sera for the H7 subtype virus identified no positive sera in any sector. Reactors to the H5 subtype virus were limited to three free-range layer chicken farms; each farm returned a single serum reactor. Follow-up investigations on these free-range farms identified evidence of historic exposure to the H5 subtype virus on one farm, and concluded that the serum reactors identified in the initial sampling round on the other two farms were non-specific (false-positive) reactions. CONCLUSIONS: The survey found no evidence of active infection with notifiable AI viruses, and provided evidence of absence of exposure to AI virus subtypes H5 and H7 in the chicken broiler, caged/barn layer, turkey broiler and pullet-rearer sectors at a between- and within-farm prevalence of 5% and 30%, respectively, with 95% confidence. The results established commercial free-range layer farms as a risk sector for exposure to notifiable AI virus.

Avian Influenza Survey in Migrating Waterfowl in Sonora, Mexico

A two-year survey was carried out on the occurrence of avian influenza in migrating birds in two estuaries of the Mexican state of Sonora, which is located within the Pacific flyway. Cloacal and oropharyngeal swabs were collected from 1262 birds, including 20 aquatic bird species from the Moroncarit and Tobari estuaries in Sonora, Mexico. Samples were tested for type A influenza (M), H5 Eurasian and North American subtypes (H5EA and H5NA respectively) and the H7 North American subtype (H7NA). Gene detection was determined by one-step real-time reverse transcription polymerase chain reaction (RRT-PCR). The results revealed that neither the highly pathogenic avian influenza virus H5 of Eurasian lineage nor H7NA were detected. The overall prevalence of avian influenza type A (M-positive) in the sampled birds was 3.6% with the vast majority in dabbling ducks (Anas species). Samples from two birds, one from a Redhead (Aythya americana) and another from a Northern Shoveler (Anas clypeata), were positive for the low-pathogenic H5 avian influenza virus of North American lineage. These findings represented documented evidence of the occurrence of avian influenza in wintering birds in the Mexican wetlands. This type of study contributes to the understanding of how viruses spread to new regions of North America and highlights the importance of surveillance for the early detection and control of potentially pathogenic strains, which could affect animal and human health.

Role of the Key Mutation in the Selective Binding of Avian and Human Influenza Hemagglutinin to Sialosides Revealed by Quantum-Mechanical Calculations

The selective binding between avian and human influenza A viral hemagglutinins (HA) subtype H3 and Neu5Acα2-3 and α2-6Gal (avian α2-3, human α2-6) is qualitatively rationalized by the fragment molecular orbital (FMO) method. We suggest a general model of analyzing protein-ligand interactions based on the electrostatic, polarization, dispersion, and desolvation components obtained from quantum-mechanical calculations at the MP2/6-31G(d) level with the polarizable continuum model of solvation. The favorable avian H3 (A/duck/Ukraine/1963)-avian α2-3 binding arises from the hydrophilic interaction between Gal-4 OH and side-chain NH(2)CO on Gln226, which is supported by the intermolecular hydrogen-bond network to the 1-COO group on Neu5Ac moiety. A substitution of Gln226Leu in the avian H3 HA1 domain increases the binding affinity to human α2-6 due to the Leu226···human α2-6 dispersion with a small entropic penalty during the complex formation. The remarkable human H3 (A/Aichi/2/1968)-human α2-6 binding is not governed by the Ser228-OH···OH-9 Neu5Ac hydrogen bond. These fragment-based chemical aspects can help design monovalent inhibitors of the influenza viral HA-sialoside binding and the simulation studies on the viral HAs-human α2-6 binding.

PB2 Residue 158 Is a Pathogenic Determinant of Pandemic H1N1 and H5 Influenza A Viruses in Mice

Influenza A viruses are human and animal pathogens that cause morbidity and mortality, which range from mild to severe. The 2009 H1N1 pandemic was caused by the emergence of a reassortant H1N1 subtype (H1N1pdm) influenza A virus containing gene segments that originally circulated in human, avian, and swine virus reservoirs. The molecular determinants of replication and pathogenesis of H1N1pdm viruses in humans and other mammals are poorly understood. Therefore, we set out to elucidate viral determinants critical to the pathogenesis of this novel reassortant using a mouse model. We found that a glutamate-to-glycine substitution at residue 158 of the PB2 gene (PB2-E158G) increased the morbidity and mortality of the parental H1N1pdm virus. Results from mini-genome replication assays in human cells and virus titration in mouse tissues demonstrated that PB2-E158G is a pathogenic determinant, because it significantly increases viral replication rates. The virus load in PB2-E158G-infected mouse lungs was 1,300-fold higher than that of the wild-type virus. Our data also show that PB2-E158G had a much stronger influence on the RNA replication and pathogenesis of H1N1pdm viruses than PB2-E627K, which is a known pathogenic determinant. Remarkably, PB2-E158G substitutions also altered the pathotypes of two avian H5 viruses in mice, indicating that this residue impacts genetically divergent influenza A viruses and suggesting that this region of PB2 could be a new antiviral target. Collectively, the data presented in this study demonstrate that PB2-E158G is a novel pathogenic determinant of influenza A viruses in the mouse model. We speculate that PB2-E158G may be important in the adaptation of avian PB2 genes to other mammals, and BLAST sequence analysis identified a naturally occurring human H1N1pdm isolate that has this substitution. Therefore, future surveillance efforts should include scrutiny of this region of PB2 because of its potential impact on pathogenesis.

Immunogenicity and protective efficacy of a DNA vaccine encoding a chimeric protein of avian influenza hemagglutinin subtype H5 fused to CD154 (CD40L) in Pekin ducks

The potential of CD154 (CD40L) as a powerful immunological adjuvant has been shown in various strategies. In this study we examine the immunogenicity and protective efficacy of a CD40-targeting avian influenza hemagglutinin (HA) subunit DNA vaccine in ducks. DNA constructs encoded the ectodomain of the HA protein of LPAI A/mallard/BC/373/2005 (H5N2) with or without fusion to the ectodomain of duck CD154. CD40-targeting significantly accelerated and enhanced humoral responses to the vector-encoded HA protein. In viral challenge experiments with A/chicken/Vietnam/14/2005 (H5N1), DNA immunization conferred partial protection against the genetically distant HPAI. The observed improved kinetics and magnitude of immune induction suggest that CD40-targeting holds promise for influenza A vaccine development.

Real-time reverse transcription-PCR assay for differentiating the Pandemic H1N1 2009 influenza virus from swine influenza viruses

Since the Pandemic H1N1 2009 (H1N1pdm) influenza virus emerged in human in 2009, H1N1pdm, classical swine H1, Eurasian avian-like H1, human-like H1 and human-like H3 swine influenza viruses have circulated in pig populations, and avian H9N2 viruses have been isolated in pigs as well. In this study, TaqMan single-step real-time reverse transcription-PCR (rtRT-PCR) assays targeting the hemagglutinin gene were developed to differentiate H1N1pdm from other genetic lineages of the H1 subtype and other subtypes of influenza viruses circulating in human and pig populations for veterinary use. H1N1pdm rtRT-PCR detected H1N1pdm RNA and did not cross-react with classical swine H1, Eurasian avian-like H1, human-like H1, human-like H3 swine and avian H9 influenza viruses RNA. Classical swine H1, Eurasian avian-like H1, human-like H1 and H3 and avian H9 rtRT-PCR were reacted exclusively with viral RNA of their respective lineages and subtypes. The results demonstrate that these assays are useful for the diagnosis of the H1N1pdm virus in both human- and animal-health-related fields.