Role of position 627 of PB2 and the multibasic cleavage site of the hemagglutinin in the virulence of H5N1 avian influenza virus in chickens and ducks

Abstract

Highly pathogenic H5N1 avian influenza viruses have caused major disease outbreaks in domestic and free-living birds with transmission to humans resulting in 59% mortality amongst 564 cases. The mutation of the amino acid at position 627 of the viral polymerase basic-2 protein (PB2) from glutamic acid (E) in avian isolates to lysine (K) in human isolates is frequently found, but it is not known if this change affects the fitness and pathogenicity of the virus in birds. We show here that horizontal transmission of A/Vietnam/1203/2004 H5N1 (VN/1203) virus in chickens and ducks was not affected by the change of K to E at PB2-627. All chickens died between 21 to 48 hours post infection (pi), while 70% of the ducks survived infection. Virus replication was detected in chickens within 12 hours pi and reached peak titers in spleen, lung and brain between 18 to 24 hours for both viruses. Viral antigen in chickens was predominantly in the endothelium, while in ducks it was present in multiple cell types, including neurons, myocardium, skeletal muscle and connective tissues. Virus replicated to a high titer in chicken thrombocytes and caused upregulation of TLR3 and several cell adhesion molecules, which may explain the rapid virus dissemination and location of viral antigen in endothelium. Virus replication in ducks reached peak values between 2 and 4 days pi in spleen, lung and brain tissues and in contrast to infection in chickens, thrombocytes were not involved. In addition, infection of chickens with low pathogenic VN/1203 caused neuropathology, with E at position PB2-627 causing significantly higher infection rates than K, indicating that it enhances virulence in chickens.

Figure 1. Virus antigen expression in tissues of infected chickens.

Chickens were infected with HP VN/1203/E (A, C, E) and LP VN/1203/E (B, D, F). Tissues were taken at 18–36 hours pi from HP infected birds and at 5–13 days pi from LP infected birds. Sections were prepared from brain (A, B), skin from the comb (B, C) and heart (E, F) and stained for the presence of viral antigen. A. Brain, showing high levels of antigen in capillaries (arrows) and in occasional neurons. B. Brain, 13 days pi, showing antigen in neurons within a focus of glial proliferation. C. Skin of comb, showing antigen in capillaries (arrows) and surrounding connective tissue within the dermis. D. Skin of comb, 13 days pi, showing viral antigen in the stratum granulosum. E. Heart, showing antigen predominantly in capillaries (arrows) and in the myocardium (indicated by arrow heads). F. Heart, 5 days pi, showing viral antigen in a single myocardial fiber. All scale bars are 100 µm.

Figure 2. Virus isolation from tissues of infected chickens.

Virus titers in thrombocytes, spleen, lung and brain samples, and from oropharyngeal (oral) and cloacal (cloaca) swabs collected from chickens infected with HP VN/1203/E and HP VN/1203/K at 12, 18 and 21 or 24 hours pi. All titrations were performed in Vero cells. Data show mean and SEM of 5–7 birds per time point for each virus.

Figure 3. Virus isolation from tissues of infected ducks.

Virus titers in spleen, lung and brain samples and from oropharyngeal (oral) and cloacal (cloaca) swabs collected from ducks infected with HP VN/1203/E and HP VN/1203/K at 1, 2, 3, 4, and 6 days pi. All titrations were performed in Vero cells. Data show mean and SEM of 6 birds per time point for each virus.