Avian Influenza Virus Subtypes H5 Research Articles

Application of real-time RT-PCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus

Avian influenza (AI) viruses are endemic in wild birds and if transmitted to poultry can cause serious economic losses. In the study of AI, the quantitation of virus shed from infected birds is valuable in pathogenesis studies and to determine the effectiveness of vaccines, and is performed routinely by cultivation of virus containing samples using embryonating chicken eggs (ECE) and expressed by 50% egg infectious dose (EID50). Although, this assay is accurate and is the standard test for infectious virus titration, the method is laborious, requires a large number of ECE, and takes at least 7 days to determine results. In this study, a one-tube hydrolysis fluorescent probe based real-time RT-PCR (RRT-PCR) was applied for the quantitation of AI virus and compared with conventional virus titration method. A strong positive correlation was observed between the amount of RNA determined by quantitative RRT-PCR and the EID50s determined by conventional methods. This RRT-PCR test was further applied in the study of competitive replication of co-infected H5 and H7 subtype viruses in chickens. Using hemagglutinin subtype specific probes, we were able to determine the amount of individual subtype virus, which could not have easily been done with conventional methods. This RRT-PCR based quantitation of AI virus, which is specific, sensitive, easy to perform, and rapid, will be useful for virological, pathogenesis, and protection studies.

Characterization of recent H5 subtype avian influenza viruses from US poultry

In the US, the isolation of H5 subtype avian influenza (AI) viruses has been uncommon in commercial chickens and turkeys, although sporadic isolations have been made from the live bird markets or its supply chain since 1986. In 2002, two different outbreaks of H5 AI occurred in commercial chicken or turkey operations. The first occurred in Texas and was identified as a H5N3 subtype AI virus. The second outbreak was caused by a H5N2 virus isolated from a turkey farm in California. In this study we analyzed recent H5 subtype AI viruses from different avian species and different sources in the US. Most recent H5 subtype isolates shared a high sequence identity and phylogenetically assorted into a separate clade from the Pennsylvania/83 lineage isolates. However, no established lineage was found within this clade and the recent H5 subtype isolates seemed to be the result of separate introductions from the wild bird reservoir. The Texas H5N3 isolate shared the lowest homology with the other recent isolates in the haemagglutinin gene and had a unique haemagglutinin cleavage site sequence of REKR/G (other recent isolates have the typical avirulent motif, RETR/G). Furthermore, this isolate had a 28 amino acid deletion in the stalk region of the neuraminidase protein, a common characteristic of chicken adapted influenza viruses, and may indicate that this virus had actually been circulating in poultry for an extended period of time before it was isolated. In agreement with genetic evidence, the Texas H5N3 isolate replicated better than other H5 isolates in experimentally infected chickens. The outbreak in Texas with a more chicken-adapted H5N3 virus underscores the importance of ongoing surveillance and control efforts regarding the H5 subtype AI virus in the US.

Application of real-time RT-PCR for the quantitation and competitive replication study of H5 and H7 subtype avian influenza virus

Avian influenza (AI) viruses are endemic in wild birds and if transmitted to poultry can cause serious economic losses. In the study of AI, the quantitation of virus shed from infected birds is valuable in pathogenesis studies and to determine the effectiveness of vaccines, and is performed routinely by cultivation of virus containing samples using embryonating chicken eggs (ECE) and expressed by 50% egg infectious dose (EID 50). Although, this assay is accurate and is the standard test for infectious virus titration, the method is laborious, requires a large number of ECE, and takes at least 7 days to determine results. In this study, a one-tube hydrolysis fluorescent probe based real-time RT-PCR (RRT-PCR) was applied for the quantitation of AI virus and compared with conventional virus titration method. A strong positive correlation was observed between the amount of RNA determined by quantitative RRT-PCR and the EID 50s determined by conventional methods. This RRT-PCR test was further applied in the study of competitive replication of co-infected H5 and H7 subtype viruses in chickens. Using hemagglutinin subtype specific probes, we were able to determine the amount of individual subtype virus, which could not have easily been done with conventional methods. This RRT-PCR based quantitation of AI virus, which is specific, sensitive, easy to perform, and rapid, will be useful for virological, pathogenesis, and protection studies.

Generation of reassortant influenza vaccines by reverse genetics that allows utilization of a DIVA (Differentiating Infected from Vaccinated Animals) strategy for the control of avian influenza

Vaccination of poultry with inactivated influenza vaccine can be an effective tool in the control of avian influenza (AI). One major concern of using inactivated vaccine is vaccine-induced antibody interference with serologic surveillance and epidemiology. In the United States, low pathogenicity H5 and H7 subtype AI viruses have caused serious economic losses in the poultry industry. Most of these viruses also have the accompanying N2 subtype and no H5N1 or H7N8 subtype AI viruses have been identified in poultry in the US. In order to allow the Differentiation of Infected from Vaccinated Animals (DIVA) while maintaining maximum efficacy of the vaccine, we generated reassortant viruses by reverse genetics that contained the same H5 and H7 hemagglutinin (HA) gene as the challenge virus, but a heterologous N1 or N8 neuraminidase (NA) gene. In vaccination-challenge experiments in 2-week-old specific pathogen free chickens, reassortant influenza vaccines (rH5N1 and rH7N8) demonstrated similar antibody profiles and comparable protection rates as vaccines prepared with parent H5N2 and H7N2 viruses. Further, we were able to differentiate the sera from infected and vaccinated birds by neuraminidase inhibition test and indirect immunofluorescent antibody assay on the basis of different antibodies elicited by their NA proteins. These results demonstrate the usefulness of a reverse genetics system for the rapid generation of reassortant AI virus that allows utilization of the DIVA strategy for the control of AI infections in poultry.

Restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host.

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (HA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 HA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 HA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague.

Infectious laryngotracheitis virus (ILTV), a member of the Alphaherpesvirinae, possesses several unique genes. One of them, UL0, encodes an abundantly expressed protein that accumulates in the nuclei of ILTV-infected cells. This study demonstrates that this protein is dispensable for in vitro virus replication and that UL0 deletion mutants exhibit only minor growth defects in cultured cells. The UL0 gene locus of ILTV was also used for insertion of foreign DNA sequences encoding enhanced GFP or haemagglutinin (HA), subtype H7, of a highly pathogenic avian influenza virus under the control of the human cytomegalovirus immediate-early gene promoter. Expression of foreign proteins was shown by (immuno)fluorescence tests and Western blot analyses. After experimental infection of chickens, UL0 deletion mutants proved to be attenuated when compared to both parental wild-type ILTV and an UL0 rescue mutant. Nevertheless, all animals immunized with UL0-negative ILTV were protected from clinical disease after subsequent infection with virulent ILTV. Furthermore, all animals immunized with HA-expressing ILTV survived a lethal challenge with H7 subtype avian influenza virus with minimal clinical signs. Thus, an UL0-negative and HA-expressing ILTV recombinant may be used as a bivalent live virus vaccine against ILT and fowl plague. Unlike inactivated influenza virus vaccines, HA-expressing ILTV recombinants should be suitable for mass application and would also permit serological discrimination between vaccinated and virus-infected animals in the field.

Rapid and sensitive detection of avian influenza virus subtype H7 using NASBA

Nucleic acid sequence-based amplification with electrochemiluminescent detection (NASBA/ECL) is an isothermal technique allowing rapid amplification and detection of specific regions of nucleic acid from a diverse range of sources. It is especially suitable for amplifying RNA. A NASBA/ECL technique has been developed allowing the detection of RNA from avian influenza virus subtype H7 derived from allantoic fluid harvested from inoculated chick embryos and from cell cultures. Degenerate amplification primers and amplicon capture probes were designed enabling the detection of low and highly pathogenic avian influenza of the H7 subtype from the Eurasian and North American lineages and the Australian sub-lineage. The NASBA/ECL technique is specific for subtype H7 and does not cross-react with other influenza subtypes or with viruses containing haemagglutinin-like genes. The assay is 10- to 100-fold more sensitive than a commercially available antigen capture immunoassay system. The NASBA/ECL assay could be used in high throughput poultry screening programmes.