A Mathematical Framework for Estimating Pathogen Transmission Fitness and Inoculum Size Using Data from a Competitive Mixtures Animal Model

We present a method to measure the relative transmissibility (“transmission fitness”) of one strain of a pathogen compared to another. The model is applied to data from “competitive mixtures” experiments in which animals are co-infected with a mixture of two strains. We observe the mixture in each animal over time and over multiple generations of transmission. We use data from influenza experiments in ferrets to demonstrate the approach. Assessment of the relative transmissibility between two strains of influenza is important in at least three contexts: 1) Within the human population antigenically novel strains of influenza arise and compete for susceptible hosts. 2) During a pandemic event, a novel sub-type of influenza competes with the existing seasonal strain(s). The unfolding epidemiological dynamics are dependent upon both the population's susceptibility profile and the inherent transmissibility of the novel strain compared to the existing strain(s). 3) Neuraminidase inhibitors (NAIs), while providing significant potential to reduce transmission of influenza, exert selective pressure on the virus and so promote the emergence of drug-resistant strains. Any adverse outcome due to selection and subsequent spread of an NAI-resistant strain is exquisitely dependent upon the transmission fitness of that strain. Measurement of the transmission fitness of two competing strains of influenza is thus of critical importance in determining the likely time-course and epidemiology of an influenza outbreak, or the potential impact of an intervention measure such as NAI distribution. The mathematical framework introduced here also provides an estimate for the size of the transmitted inoculum. We demonstrate the framework's behaviour using data from ferret transmission studies, and through simulation suggest how to optimise experimental design for assessment of transmissibility. The method introduced here for assessment of mixed transmission events has applicability beyond influenza, to other viral and bacterial pathogens

Contact transmission of influenza virus between ferrets imposes a looser bottleneck than respiratory droplet transmission allowing propagation of antiviral resistance

Influenza viruses cause annual seasonal epidemics and occasional pandemics. It is important to elucidate the stringency of bottlenecks during transmission to shed light on mechanisms that underlie the evolution and propagation of antigenic drift, host range switching or drug resistance. The virus spreads between people by different routes, including through the air in droplets and aerosols, and by direct contact. By housing ferrets under different conditions, it is possible to mimic various routes of transmission. Here, we inoculated donor animals with a mixture of two viruses whose genomes differed by one or two reverse engineered synonymous mutations, and measured the transmission of the mixture to exposed sentinel animals. Transmission through the air imposed a tight bottleneck since most recipient animals became infected by only one virus. In contrast, a direct contact transmission chain propagated a mixture of viruses suggesting the dose transferred by this route was higher. From animals with a mixed infection of viruses that were resistant and sensitive to the antiviral drug oseltamivir, resistance was propagated through contact transmission but not by air. These data imply that transmission events with a looser bottleneck can propagate minority variants and may be an important route for influenza evolution

Multidrug Resistant 2009 A/H1N1 Influenza Clinical Isolate with a Neuraminidase I223R Mutation Retains Its Virulence and Transmissibility in Ferrets

Only two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that influenza virus becomes resistant to these antiviral drugs and spreads in the human population. The 2009 pandemic A/H1N1 influenza virus is naturally resistant to adamantanes. Recently a novel neuraminidase I223R mutation was identified in an A/H1N1 virus showing cross-resistance to the neuraminidase inhibitors oseltamivir, zanamivir and peramivir. However, the ability of this virus to cause disease and spread in the human population is unknown. Therefore, this clinical isolate (NL/2631-R223) was compared with a well-characterized reference virus (NL/602). In vitro experiments showed that NL/2631-I223R replicated as well as NL/602 in MDCK cells. In a ferret pathogenesis model, body weight loss was similar in animals inoculated with NL/2631-R223 or NL/602. In addition, pulmonary lesions were similar at day 4 post inoculation. However, at day 7 post inoculation, NL/2631-R223 caused milder pulmonary lesions and degree of alveolitis than NL/602. This indicated that the mutant virus was less pathogenic. Both NL/2631-R223 and a recombinant virus with a single I223R change (recNL/602-I223R), transmitted among ferrets by aerosols, despite observed attenuation of recNL/602-I223R in vitro. In conclusion, the I223R mutated virus isolate has comparable replicative ability and transmissibility, but lower pathogenicity than the reference virus based on these in vivo studies. This implies that the 2009 pandemic influenza A/H1N1 virus subtype with an isoleucine to arginine change at position 223 in the neuraminidase has the potential to spread in the human population. It is important to be vigilant for this mutation in influenza surveillance and to continue efforts to increase the arsenal of antiviral drugs to combat influenza

The Molecular Basis of Fitness and Transmissibility of Neuraminidase Inhibitor Resistant Influenza A Viruses

Neuraminidase (NA) inhibitors including oral oseltamivir and inhaled zanamivir are among the first line of defense against influenza virus infection. Development of resistance to NA inhibitors is a huge drawback for limited options for the control of influenza. During the first decade of NA inhibitor use, the detection rates of resistance to both NA inhibitors had remained low in circulating influenza viruses. However, the 2008~2009 season was marked by a radical increase of prevalence of oseltamvir resistance from \u3c1% to \u3e90% in worldwide surveillance in less than a year. The resistance was solely linked to NA H275Y variants of seasonal H1N1 viruses, and they are referred as the naturally resistant viruses. A big question remains open about what fundamental molecular changes in the seasonal H1N1 viruses led to the surge of the naturally resistant viruses. When this question remained pending, a novel swine-origin H1N1 influenza virus emerged in Mexico at April 2009, soon spread worldwide replacing the seasonal influenza viruses including the naturally resistant viruses, and marked 2009 with the first influenza pandemic of the 21st century. With sustainably increased worldwide use of NA inhibitors especially oral oseltamivir during the pandemic, the oseltamivir-resistant variants carrying H275Y NA mutation were isolated at low incidence from individuals receiving oseltamivir treatment and a few community clusters. In view of the high prevalence of naturally resistant seasonal H1N1 viruses in the immediate preceding season, there was an urgent need to characterize the transmissibility and fitness of oseltamivir-resistant pandemic H1N1/2009 viruses, although the resistance rates have remained low so far. We first addressed the urgent question about pandemic viruses by investigating the transmissibility of a closely matched pair of pandemic H1N1/2009 clinical isolates, which only differed at the H275Y NA mutation in their genome, in the ferret model. We found that the H275Y NA mutant H1N1/2009 virus was not transmitted efficiently in ferrets via respiratory droplets, while it retained efficient transmission via direct contact. The wild-type H1N1/2009 virus was efficiently transmitted via both routes. The wild-type and the mutant viruses appeared to cause a similar disease course in ferrets without apparent attenuation of clinical signs. In the growth competition in a ferret, the H275Y mutant virus showed less growth capability than the wild-type virus. The NA of the H275Y mutant virus showed reduced substrate-binding affinity and catalytic activity in vitro and delayed initial growth in MDCK and MDCK-SIAT1 cells. These findings may in part explain its less efficient transmission. The fact that the oseltamivir-resistant H1N1/2009 virus retained efficient transmission through direct contact underlines the necessity of continuous monitoring of drug resistance and characterization of more NA inhibitor-resistant variants of the pandemic H1N1 viruses. We also sought to resolve the pending question about the naturally resistant seasonal H1N1 viruses by investigating the changes of different seasonal H1N1 viruses in terms of NA genetics, NA proteins attributes and virus fitness. We found that during the seasonal H1N1 virus evolution, two genetically diverged lineages of H1N1 viruses were circulating at different times. The NA protein phenotypes of the two lineages were naturally distinct in the levels of protein expression and enzyme affinity, and accordingly, the H275Y NA mutation had differential effects on the NA proteins and virus fitness of the two lineages. The new lineage NA proteins were inherently higher in protein expression and enzyme affinity than the old lineage NA proteins and thus were able to tolerate the negative effects of the H275Y mutation with a marginal loss of enzyme activity. As a result, the H275Y mutant H1N1 viruses of the new lineage had virus fitness equivalent to the wild-type viruses and were able to continue circulating, becoming the naturally resistant viruses. Further study revealed that 4 different amino acid substitutions played different roles in maintaining high protein expression and enzyme affinity of the new lineage NA proteins; the timeline of the sequential acquisition of the 4 substitutions was consistent with the timeline of emergence of the naturally resistant H1N1 viruses. The identified NA tolerance to the H275Y mutation in the naturally resistant seasonal H1N1 viruses also had implication on the virus fitness of the H275Y mutant H1N1/2009 viruses, as well as on the continuing surveillance monitoring of circulating pandemic H1N1 viruses. Overall, both studies investigated in vitro and in vivo fitness of H275Y mutant H1N1 viruses relative to their respective wild-type viruses, which were circulating in human beings at different times. These studies correlated the viral fitness of the H275Y mutant viruses with the NA tolerance to the H275Y mutation at protein level, and revealed that the NA tolerance to the H275Y mutation was the molecular determinant of fitness of H275Y mutant H1N1 viruses. The studies have implications on surveillance monitoring of the NA inhibitor resistance in circulating influenza viruses, which underlines the necessity of continuous monitoring of drug resistance incidence, as well as potential genetic and phenotypic changes of constantly evolving influenza viruses

Pathogenesis of influenza in the Ferret Model : a basis for improved intervention

Although clinical disease and associated lesions of the respiratory tract due to IAV infections are well known, the exact pathogenesis of acute respiratory distress syndrome (ARDS) is not yet fully understood. ARDS is a fatal complication of influenza virus infection, and accounts for many influenza-­‐related mortalities. At the starting point of this thesis, the mechanism of pulmonary oedema formation, as one of the hallmarks