The Asian H5N1 highly pathogenic avian influenza (HPAI) viruses have been increasing in pathogenicity in diverse avian species since 1996 and are now widespread in Asian, European, and African countries. To better understand the basis of the increased pathogenicity of recent Asian H5N1 HPAI viruses in chickens, we compared the fevers and mean death times (MDTs) of chickens infected with the Asian H5N1 A/chicken/Yamaguchi/7/04 (CkYM7) strain with those infected with the H5N1 Duck/Yokohama/aq10/03 (DkYK10) strain, using a wireless thermosensor. Asian H5N1 CkYM7 caused peracute death in chickens before fever could be induced, whereas DkYK10 virus induced high fevers and had a long MDT. Real-time PCR analyses of cytokine mRNA expressions showed that CkYM7 quickly induced antiviral and proinflammatory cytokine mRNA expressions at 24 h postinfection (hpi) that suddenly decreased at 32 hpi. In contrast, these cytokine mRNA expressions increased at 24 hpi in the DkYK10 group, but decreased from 48 hpi onward to levels similar to those resulting from infection with the low-pathogenicity H5N2 A/chicken/Ibaraki/1/2004 strain. Sequential titrations of viruses in lungs, spleens, and kidneys demonstrated that CkYM7 replicated rapidly and efficiently in infected chickens and that the viral titers were more than twofold higher than those of DkYK10. CkYM7 preferentially and efficiently replicated in macrophages and vascular endothelial cells, while DkYK10 grew moderately in macrophages. These results indicate that the increased pathogenicity in chickens of the recent Asian H5N1 HPAI viruses may be associated with extremely rapid and high replication of the virus in macrophages and vascular endothelial cells, which resulted in disruption of the thermoregulation system and innate immune responses.
Avian influenza (AI) virus belongs to the family Orthomyxoviridae and is classified into low-pathogenicity (LP) and highpathogenicity (HP) pathotypes based on a pathogenicity test for chickens. The HPAI virus has over 75% mortality in chickens and can have devastating economic consequences, which can be controlled by a World Organization for Animal Health stamping-out policy, in the poultry industry. The HPAI viruses identified to date are only of the H5 and H7 subtypes and have multiple basic amino acid residues at the hemagglutinin (HA) cleavage site. Of the HPAI viruses, the H5N1 HPAI virus that has continued to circulate in poultry in East Asia since 1996 has been shown to be extremely virulent in chickens (47) and a serious threat to human health. The H5N1 virus has caused over 400 human infections in 15 countries and has a mortality rate of more than 50%.Molecular mechanisms for adaptation of AI viruses from natural reservoirs to new hosts are important for understanding the evolution of influenza viruses. The binding property of hemagglutinin (HA) proteins to avian or mammalian sialic acid receptors (␣2-3 or ␣2-6, respectively) is a first step in overcoming the interspecies barrier. The NS1 protein plays an important role in countering host cell antiviral cytokines or the initial host immune responses of chickens (22, 43). Recently, it was shown that amino acids at position 627 (8, 11) and 701 (21, 46) in polymerase subunit PB2 and 97, 349, and 550 in polymerase subunit PA (40, 45) may play important roles in the adaptation of H5N1 HPAI viruses from birds to mammals. In contrast, the molecular basis of the pathogenicity of AI viruses in chickens on the particle surface HA and neuraminidase (NA) proteins has been intensely investigated. The acquisition of polybasic amino acids at the HA cleavage site is a main determinant permitting the systemic replication of AI viruses in chickens, and the glycosylation patterns of HA molecules influence the accessibility of proteinases to the HA cleavage site (15,35,42). Amino acids in proximity to the HA receptor binding site affect the pathogenicity of the virus in chickens (17). The NA protein is implicated in the release of influenza virus from cells by removing sialic acid residues from the cellderived glycoproteins (33, 47); virus particles with low NA activity cannot be released efficiently from infected cells (23,27). A functional association between the HA and NA proteins has been suggested to directly affect pathogenicity (17,28). Interestingly, recent studies have shown that polymerase subunits (PB1, PB2, and PA) and nucleoprotein (NP) also contribute to pathogenicity in avian species, as has been shown for
Serious concern about the worldwide transmission of the Asian H5N1 highly pathogenic (HP) avian influenza (AI) virus by migratory birds surrounds the importance of the AI global surveillance in wild aquatic birds and underscores the requirement for a reliable subtyping method of AI viruses. PCR is advantageous due to its simplicity, lower cross-reactivity, and unlimited reagent supply. Currently, the only available hemagglutinin (HA) subtyping primer set that can subtype H1 through H15 is not fully evaluated and, since it only targets HA1, is unavailable for molecular pathotyping of AI viruses. Our preliminary experiments found that these primer sets were cross-reactive and missed some recent AI viruses. In this study, we developed new primer sets against HA cleavage sites for subtyping H1 to H15 genes and for molecular pathotyping. Our primer sets were subtype specific and detected 99% of previously identified HA genes ( . These results indicate that our primers are useful not only for HA subtyping but also for molecular pathotyping of both previous and recent AI viruses. These advancements will enable general diagnostic laboratories to subtype AI viruses for the surveillance in wild aquatic birds.
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