Identification of viral factors and host responses associated with the pathogenesis of influenza A (H7N9) viruses

Abstract

The avian influenza H7N9 virus that emerged in China in 2013 has repeatedly caused human infections and is a threat to public health due to the severe illness in a majority of patients. However, a wide range of clinical outcomes, including asymptomatic infections, have been observed, which may be related to the characteristics of the virus itself and the host response to the virus. Identifying the viral factors and host responses that may underlie mild or severe infections might help explain the pathogenesis of the H7N9 virus in mammals and lead to options for treatment. This thesis examines the viral and host response factors using the virulent H7N9 human isolate A/Shanghai/1/2013 (SH1) and the mild A/Shanghai/05/2013 (SH5). Experiments using recombinant viruses of these two strains in a mouse model were conducted to identify the virulence-contributing segments and the key amino acids involved. Transcriptional profiles of mouse lungs infected by H7N9 viruses of different virulence levels were characterized to investigate virulence-related host responses. Infections of mice with avian origin H5N1 viruses that cause similar pathology to the H7N9 viruses were used for comparative transcriptional profiling to examine the general and subtype- specific host response. Thirty-eight reassortant viruses were constructed to investigate the key viral factors in mice. Although concurrent exchange of the HA and NA, NA and M segments between SH1 and SH5 viruses posed some impact on virulence, independent replacement of the PB2 and PB1 segments influenced the virulence most significantly. The mammalian adaptation signature PB2-E627K was found to be the major viral factor responsible for the high polymerase activity in mammalian cells, high virus replication and virulence in mice of SH1 virus, but roles of PB2-E191K and PB1-L598M mutations were also detected. Transcriptional profiling of infection with H7N9 viruses revealed uncontrolled inflammatory responses (cytokine storm, apoptosis), severe lung tissue damage (deposit of matrix metalloproteinase and collagen), impaired tissue repair (inhibited lipid metabolism and cellular development), and impaired B cell response in virulent infections. The expression pattern of mucin genes was shown to be related to disease outcome and may serve as an indicator of disease severity after influenza virus infection. Enhanced inflammation (including oxidative stress and consequent damage to ion transport functions) was common in both virulent H7N9 and H5N1 virus infections. Possible subtype effects related to stronger activation of protein ubiquitination and natural killer cell signalling pathways were seen in virulent H7N9 and H5N1 virus infections, respectively. In particular, the PB2-627K mutation in H5N1 virus caused stronger NK cell recruitment that may correlate with the brain tropism of virus. This thesis has provided a systematic study on the contribution of each segment, and several amino acid mutations, to the virulence of H7N9 viruses in a mammalian model. It has characterized host responses during the progression of disease and shown the role of strong activation of the immune response in severe disease. These findings will help surveillance studies identify potentially more pathogenic viruses and open opportunities for the development of therapeutic strategies to combat H7N9 and other influenza virus infections.