H5N1 influenza virus pathogenesis in genetically diverse mice is mediated at the level of viral load

Abstract

The genotype of the host is one of several factors involved in the pathogenesis of an infectious disease and may be a key parameter in the epidemiology of highly pathogenic H5N1 influenza virus infection in humans. Gene polymorphisms may affect the viral replication rate or alter the host's immune response to the virus. In humans, it is unclear which aspect dictates the severity of H5N1 virus disease. To identify the mechanism underlying differential responses to H5N1 virus infection in a genetically diverse population, we assessed the host responses and lung viral loads in 21 inbred mouse strains upon intranasal inoculation with A/Hong Kong/213/03 (H5N1). Resistant mouse strains survived large inocula while susceptible strains succumbed to infection with 1,000- to 10,000-fold-lower doses. Quantitative analysis of the viral load after inoculation with an intermediate dose found significant associations with lethality as early as 2 days postinoculation, earlier than any other disease indicator. The increased viral titers in the highly susceptible strains mediated a hyperinflamed environment, indicated by the distinct expression profiles and increased production of inflammatory mediators on day 3. Supporting the hypothesis that viral load rather than an inappropriate response to the virus was the key severity-determining factor, we performed quantitative real-time PCR measuring the cytokine/viral RNA ratio. No significant differences between susceptible and resistant mouse strains were detected, confirming that it is the host genetic component controlling viral load, and therefore replication dynamics, that is primarily responsible for a host's susceptibility to a given H5N1 virus.

Importance: Highly pathogenic H5N1 influenza virus has circulated in Southeast Asia since 2003 but has been confirmed in relatively few individuals. It has been postulated that host genetic polymorphisms increase the susceptibility to infection and severe disease. The mechanisms and host proteins affected during severe disease are unknown. Inbred mouse strains vary considerably in their ability to resist H5N1 virus and were used to identify the primary mechanism determining disease severity. After inoculation with H5N1, resistant mouse strains had reduced amounts of virus in their lungs, which subsequently resulted in lower production of proinflammatory mediators and less pathology. We therefore conclude that the host genetic component controlling disease severity is primarily influencing viral replication. This is an important concept, as it emphasizes the need to limit virus replication through antiviral therapies and it shows that the hyperinflammatory environment is simply a reflection of more viral genetic material inducing a response.

FIG 1

Pathogenic profile of 21 inbred mouse strains inoculated with highly pathogenic H5N1 influenza A virus. The 50% mouse lethal dose (MLD₅₀) of A/Hong Kong/213/03 virus is shown. A dose of 10⁴ EID₅₀ (dotted line) was set to distinguish influenza virus-resistant from influenza virus-susceptible mouse strains and used throughout the work to assess biological parameters associated with severity of disease and mortality.

FIG 2

The hematopoietic component does not confer resistance in susceptible strains. Chimeric DBA/2_S mice containing bone marrow from resistant (C57BL/6_R or BALB/c_R) or susceptible (DBA/2_S) mice were inoculated with 10⁴ EID₅₀ (A) or 10³ EID₅₀ (B) of A/Hong Kong/213/03 virus and monitored for morbidity and mortality for 21 days.

FIG 3

Principal component analysis identifies the gene expression profile associated with severity of H5N1 virus disease. Principal component analysis using ~26,000 probes in lung tissue RNA of DBA/2_S, 129/SvIm_S, A/J_S, SM_R, C57BL/6_R, and BALB/c_R mice prior to infection (control) and on days 1, 3, and 7 after inoculation with 10⁴ EID₅₀ of A/Hong Kong/213/03. Each dot represents the RNA expression profile of a single mouse. Colored circles indicate the three clusters of highly susceptible (red), intermediate susceptible (blue), and resistant (green) mouse strains. The x, y, and z axes correspond to principal components 1, 2, and 3, respectively.

FIG 4

Elevated production of proinflammatory mediators in susceptible mouse strains after inoculation with H5N1 virus. Cytokine concentrations (pg/ml) were measured in homogenized lung tissues of DBA/2_S, 129/SvIm_S, A/J_S, SM_R, C57BL/6_R, and BALB/c_R mice 3 days after inoculation with highly pathogenic A/Hong Kong/213/03 H5N1 virus. Bars represent mean cytokine production levels + SEMs. Statistical significance (P < 0.01) between the six mouse strains is represented by letters above each column, with different letters signifying distinct statistical groups.

FIG 5

Viral load determines proinflammatory cytokine production in genetically diverse mice. (A) Higher viral load (∆∆C_T value) in susceptible mouse strains (DBA/2_S, 129SvIm_S, and A/J_S) than in the resistant strains C57BL/6_R and BALB/c_R 1 and 3 days postinoculation (dpi) with 10⁴ EID₅₀ of H5N1 virus. Dots indicate individual mice, and the average values + SEMs are indicated by the solid bars. (B) Resistant mouse strains (SM_R, C57BL/6_R, and BALB/c_R) more effectively limit virus replication between 1 and 3 dpi than do susceptible strains. Bars represent average fold increases in viral RNA + SEMs. (C) Higher viral load in susceptible mouse strains plus SM_R triggers an increase in production of proinflammatory mediators CCL2, IFN-β1, and TNF-α at 3 dpi (gray bars; P < 0.01) but not at 1 dpi (black bars; P > 0.05). Bars represent average ∆∆C_T values + SEMs.