H5 Hemagglutinin Subtypes Research Articles

Highly sensitive detection of influenza virus with SERS aptasensor.

Highly sensitive and rapid technology of surface enhanced Raman scattering (SERS) was applied to create aptasensors for influenza virus detection. SERS achieves 106−109 times signal amplification, yielding excellent sensitivity, whereas aptamers to hemagglutinin provide a specific recognition of the influenza virus. Aptamer RHA0385 was demonstrated to have essentially broad strain-specificity toward both recombinant hemagglutinins and the whole viruses. To achieve high sensitivity, a sandwich of primary aptamers, influenza virus and secondary aptamers was assembled. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labelled with Raman-active molecules. The signal was affected by the concentration of both primary and secondary aptamers. The limit of detection was as low as 1 · 10−4 hemagglutination units per probe as tested for the H3N2 virus (A/England/42/72). Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes. Therefore, the aptasensors could be applied for fast and low-cost strain-independent determination of influenza viruses.

Sequential DNA immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin

Vaccines against avian influenza are mostly based on hemagglutinin (HA), which is the main antigen of this virus and a target for neutralizing antibodies. Traditional vaccines are known to be poorly efficient against newly emerging strains, which is an increasing worldwide problem for human health and for the poultry industry. As demonstrated by research and clinical data, sequential exposure to divergent influenza HAs can boost induction of universal antibodies which recognize conserved epitopes. In this work, we have performed sequential immunization of laying hens using monovalent or bivalent compositions of DNA vaccines encoding HAs from distant groups 1 and 2 (H5, H1, and H3 subtypes, respectively). This strategy gave promising results, as it led to induction of polyclonal antibodies against HAs from both groups. These polyclonal antibodies showed cross-reactivity between different HA strains in ELISA, especially when bivalent formulations were used for immunization of birds. However, cross-reactivity of antibodies induced against H3 and H5 HA subtypes was rather limited against each other after homologous immunization. Using a cocktail of HA sequences and/or sequential DNA vaccination with different strains presents a good strategy to overcome the limited effectiveness of vaccines and induce broader immunity against avian influenza. Such a strategy could be adapted for vaccinating laying hens or parental flocks of different groups of poultry.

Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

ABSTRACTHighly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.

Development and applications of universal H7 subtype-specific antibodies for the analysis of influenza H7N9 vaccines

H7N9 is a newly emerged avian influenza virus with a relatively high mortality rate in humans. At this time, there is no licensed vaccine for human protection. Development of analytical tools for H7N9 vaccine could facilitate vaccine development. Here, a universally conserved epitope in all H7 hemagglutinin (HA) sequences was identified through comprehensive bioinformatics analyses. The peptide epitope, RSGSSFYAEMK, (aa positions 149 to 159), is located on the head of the HA molecule. Antibodies generated against this universal H7 epitope were remarkably specific against H7 viral sequence with no detectable cross-reactivity to other HA subtypes. A new immunoblotting assay based on the universal H7 antibody was developed and compared with the traditional single radial immunodiffusion assay (SRID) for potency analyses of candidate H7N9 vaccines. This new assay was more sensitive and rapid compared to SRID. In addition to statistically acceptable precision and reproducibility, the new assay differs from many other alternative potency assays for influenza vaccine in that it is potentially stability-indicating, which is an important requirement for industry vaccine stability studies analyses. Furthermore, the robustness of this new assay was demonstrated by the quantitative determination of HA content in four H7N9 vaccines (split or inactivated) from different manufacturers.

Canada's inter‐agency wild bird influenza survey

Canada's Inter-agency Wild Bird Influenza Survey, which started in 2005, and is a collaborative effort among federal, provincial and territorial government agencies as well as non-governmental organizations and academic institutions, has two components: (i) a seasonal survey of live wild waterfowl species from selected geographic regions across Canada (to expand our understanding of the avian influenza viruses circulating in wild bird populations); and (ii) an ongoing survey of birds found dead that are submitted to a regional diagnostic laboratory (to enhance detection of highly pathogenic avian influenza strains). Combined swabs from the cloaca and oropharynx collected from each bird are screened using a real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) that targets a unique segment of the influenza A M1 gene. If the M1 result is positive or inconclusive, RRT-PCR for gene segments of the H5 and H7 hemagglutinin subtypes are performed. All samples that are RRT-PCR positive for H5 or H7 are sent immediately for test confirmation and further characterization. All field and laboratory data are entered into a database developed and maintained by the Canadian Cooperative Wildlife Health Centre. Since the survey commenced in 2005, on average, 30% of all live ducks sampled, 5% of other species of live birds and 3% of birds found dead have tested positive for avian influenza, all of North American lineage and of low pathogenicity.

Development of Real Time RT-PCR Assays for Detection of Type A Influenza Virus and for Subtyping of Avian H5 and H7 Hemagglutinin Subtypes

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

A Reverse transcription-PCR assay for detection of type A influenza virus and differentiation of avian H7 subtype

Avian influenza virus (AIV) infection is a major cause of influenza mortality in birds and can cause human mortality and morbidity. Although the risk of infection with avian influenza virus (AIV) is generally low for most people, the pathogenic virus can cross the species barrier and acquires the ability to infect and be transmitted among the human population; therefore the rapid identification of the virus is of important clinical and epidemiological implication. A reverse transcriptase-polymerase chain reaction (RT-PCR) was optimized for the detection of type A influenza virus. The assay differentiates avian H7 hemagglutinin subtypes. Two sets of specific oligonucleotide primers were used in this test for type A influenza virus and H7 heamagglutinin subtypes. The RT-PCR DNA products were visualized by gel electrophoresis and consisted of fragments of 98 bp for H7 hemagglutinin subtypes and 101 bp for type A influenza virus. The common set of primers for type A influenza virus were able to amplify a 101 bp DNA band for any of the other subtypes of influenza A virus. The RT-PCR assay developed in this study was found to be sensitive and specific. No specific amplification bands of the same sizes (98 bp) could be amplified for RNA of other influenza hemagglutinin subtypes, specific amplification bands of type A influenza (101 bp) for Influenza B, C, or other viral or bacterial pathogens was not tested in this study. Key word:. Influenza A RT-PCR Hemagglutinin H7 Subtyping

Engineered viral vaccine constructs with dual specificity: Avian influenza and Newcastle disease

Avian influenza viruses of the H5 and H7 hemagglutinin subtypes, and Newcastle disease virus (NDV), are important pathogens in poultry worldwide. Specifically, the highly pathogenic H5N1 avian influenza virus is a particular threat because it has now occurred in more than 40 countries on several continents. Inasmuch as most chickens worldwide are vaccinated with a live NDV vaccine, we embarked on the development of vaccine prototypes that would have dual specificity and would allow a single immunization against both avian influenza and Newcastle disease. Using reverse genetics, we constructed a chimeric avian influenza virus that expressed the ectodomain of the hemagglutinin-neuraminidase gene of NDV instead of the neuraminidase protein of the H5N1 avian influenza virus. Our second approach to creating a bivalent vaccine was based on expressing the ectodomain of an H7 avian influenza virus hemagglutinin in a fusogenic and attenuated NDV background. The insertion into the NDV genome of the foreign gene (containing only its ectodomain, with the transmembrane and cytoplasmic domains derived from the F protein of NDV) resulted in a chimeric virus with enhanced incorporation of the foreign protein into virus particles. A single immunization of chickens with this improved vaccine prototype virus induced not only a 90% protection against an H7N7 highly pathogenic avian influenza virus, but also complete immunity against a highly virulent NDV. We propose that chimeric constructs should be developed for convenient, affordable, and effective vaccination against avian influenza and Newcastle disease in chickens and other poultry.

Application and Evaluation of RT-PCR—ELISA for the Nucleoprotein and RT-PCR for Detection of Low-Pathogenic H5 and H7 Subtypes of Avian Influenza Virus

Three 1-tube Reverse Transcriptase Polymerase Chain Reactions (RT-PCR) directed against the genes encoding the nucleoprotein (NP) and the H5 and H7 hemagglutinin (HA) gene, respectively, were used for detection of avian influenza virus (AIV) in various specimens. A total of 1,040 samples originating from chickens experimentally infected with 2 different low pathogenic avian influenza viruses, from domestic ducks and from wild aquatic birds were examined. The outcome of 1) the universal AIV RT-PCR including a PCR-enzyme-linked immunosorbent assay (ELISA) procedure directed against NP (NP RT-PCR-ELISA) and 2) the subtype specific RT-PCR for H5 and H7 were compared to the results obtained by inoculation of the same specimens into the allantoic cavity of embryonated specific pathogen free (SPF) hen's eggs. Using inoculation in SPF fowl eggs as standard the sensitivity of the NP RT-PCR-ELISA and the RT-PCR for H5 or H7 was 91% and 94%, and the corresponding specificity 98% and 96%. In comparison with inoculation into eggs an additional of 9 samples were positive by NP RT-PCR-ELISA and 13 samples were positive by RT-PCR for one of the HA subtypes. Hence, the 3 RT-PCR procedures described are fast, sensitive and specific for detecting AIV and subtyping H5 and H7 and they are obvious alternatives when testing large numbers of samples.

Characterization of avian influenza virus isolates submitted to the National Centre for Foreign Animal Disease between 1997 and 2001.

The National Centre for Foreign Animal Disease (NCFAD) in Winnipeg, Manitoba, is the Canadian Food Inspection Agency's (CFIA) newest high biocontainment laboratory. One of the functions of the NCFAD is to serve as a national reference laboratory for avian influenza. Between 1997 and 2001, 15 avian influenza virus isolates were characterized. These isolates originated from domestic poultry, imported caged birds held in quarantine, and wild birds. Diagnostic specimens were submitted to the NCFAD by CFIA field veterinarians, provincial veterinary diagnostic laboratories, and veterinary colleges. Characterization of isolates included the determination of H and N subtypes: H1, H6, H7, and H10 subtypes were isolated from domestic poultry; H3, H4, and three H13 viruses were isolated from water fowl, and six H3 viruses were isolated from caged birds being held in import quarantine. Selected isolates were characterized with respect to their pathogenic potential by intravenous inoculation of 4-to-6-wk-old chickens. A molecular-based protocol was used to assess the pathogenicity of one H7 isolate. During this period, work was also carried out toward validating our molecular pathotyping protocol for avian influenza viruses with H5 and H7 hemagglutinin subtypes.

Tissue Tropism and Replicative Properties of Waterfowl-Origin Influenza Viruses in Chickens

Waterfowl-origin influenza (WFOI) viruses were evaluated for their tissue tropism and replicative properties in chickens. The 14 WFOI isolates used in this study represented 13 different hemagglutinin-neuraminidase combinations recovered during 1987 and 1988 and included isolates possessing the H5 and H7 hemagglutinin subtypes and one isolate possessing the H5N2 combination. Following intravenous challenge, the frequencies of virus recovery within individual experiments were generally higher for the lower digestive tract and kidney samples. Virus titers ranged up to 10(8.5) mean embryo infective doses per gram of kidney tissue in clinically normal chickens. Differences in frequencies of virus recovery and virus titers in tissues indicated that some of these uniformly nonpathogenic and low-pathogenicity WFOI virus isolates replicated more extensively in chickens than did others. This enhanced ability to replicate in chickens should be further evaluated as a potential factor associated with the threat WFOI viruses present to poultry.