Genetic and phenotypic characterization of a clade 2.3.2.1b influenza A (H5N1) virus isolated from a patient with encephalitis

Abstract

Highly pathogenic avian influenza H5N1 viruses have affected poultry and other birds worldwide and have transmitted zoonotically to humans. The overall mortality of H5N1 disease was around 60%. It has been suggested that genetic mutation(s) in the haemagglutinin (HA) and the basic polymerase 2 (PB2) proteins are key determinants of transmission and pathogenicity when inter-species transmission occurs from avian to human hosts. The mechanisms by which avian H5N1 viruses cause high disease severity in mammals is still not clear.

In 2012, a 2-year-old boy with highly pathogenic avian influenza A(H5N1) virus infection (henceforth designated as H5N1/5923) with minimal respiratory symptoms developed encephalitis complicated by obstructive hydrocephalus. Genetically, this virus belonged to H5N1 clade 2.3.2.1b and had acquired the mammalian adaptation mutation PB2 Q591K. In this study, we aimed to understand the characteristics of this H5N1 virus in adapting to a mammalian host. We used clinical specimens collected during the course of the illness and genetically and phenotypically characterized the virus isolate obtained using experimental models in vitro and in vivo.

We detected virus in non-respiratory specimens including cerebrospinal fluid (CSF), brain tissue and plasma/serum. Although patients infected with H5N1 viruses rarely had detectable viral RNA in the respiratory tract for longer than 3 weeks, we found viral RNA in the nasopharyngeal aspirate (NPA) specimens of our patient even on day 30 after symptom onset.

To study viral intra-host genetic diversity (also termed virus “quasispecies”), we identified HA genetic variations by using Sanger sequencing of genetically cloned nucleic acid from the original clinical specimen. We investigated whether these mutations in the HA contributed to the switching of receptor binding affinity to human/mammalian sialic acid receptors with alpha 2-6 linked glycans using the haemagglutination assay and saturation transfer difference Nuclear Magnetic Resonance studies (STD-NMR) and found no evidence of adaption to mammalian receptors.

Dissemination of H5N1 virus beyond the respiratory tract was reported to be facilitated by the presence of amino acid substitutions in PB2 gene. The H5N1/5923 virus had an unusual amino acid substitution PB2-591K. Our results showed that the polymerase activity of PB2-591K had a phenotype comparable with a previously reported mammalian adaptation mutation PB2-627K. We demonstrated that the PB2-Q591K can lead to the virus spread into the brain in experimentally infected mice. In structural analysis, we found PB2-591K is associated with a greater positive charge on the binding interface of the PB2 molecule, comparable with the well-known mammalian adaptation mutation PB2-627K and is thus compatible with higher polymerase activity.

Understanding the viral genetic determinants associated with severe disease such as encephalitis will help elucidate the pathogenesis of human H5N1 disease.