TaqMan real time RT-PCR assays for detecting ferret innate and adaptive immune responses

AbstractThe ferret is an excellent model for many human infectious diseases including influenza, SARS-CoV, henipavirus and pneumococcal infections. The ferret is also used to study cystic fibrosis and various cancers, as well as reproductive biology and physiology. However, the range of reagents available to measure the ferret immune response is very limited. To address this deficiency, high-throughput real time RT-PCR TaqMan assays were developed to measure the expression of fifteen immune mediators associated with the innate and adaptive immune responses (IFNα, IFNβ, IFNγ, IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL17, Granzyme A, MCP1, TNFα), as well as four endogenous housekeeping genes (ATF4, HPRT, GAPDH, L32). These assays have been optimized to maximize reaction efficiency, reduce the amount of sample required (down to 1ng RNA per real time RT-PCR reaction) and to select the most appropriate housekeeping genes. Using these assays, the expression of each of the tested genes could be detected in ferret lymph node cells stimulated with mitogens or infected with influenza virus in vitro. These new tools will allow a more comprehensive analysis of the ferret immune responses following infection or in other disease states

Pathogenesis, humoral immune responses and transmission between co-housed animals in a ferret model of human RSV infection

Small animal models have been used to obtain many insights regarding the pathogenesis and immune responses induced following infection with human respiratory syncytial virus (hRSV). Amongst those described to date, infections in cotton rats, mice, guinea pigs, chinchillas and Syrian hamsters with hRSV strains Long and/or A2 have been well characterised, although clinical isolates have also been examined. Ferrets are also susceptible to hRSV infection but the pathogenesis and immune responses elicited following infection have not been well characterised. Herein, we describe the infection of adult ferrets with hRSV Long or A2 via the intranasal route and characterised virus replication, as well as cytokine induction, in the upper and lower airways. Virus replication and cytokine induction during the acute phase of infection (days 0-15 post-infection) were similar between the two strains and both elicited high levels of F glycoprotein-specific binding and neutralising antibodies following virus clearance (days 16-22 post-infection). Importantly, we demonstrate transmission from experimentally infected donor ferrets to co-housed naïve recipients and have characterised virus replication and cytokine induction in the upper airways of infected contact animals. Together, these studies provide a direct comparison of the pathogenesis of hRSV Long and A2 in ferrets and highlight the potential of this animal model to study serological responses and examine interventions that limit transmission of hRSV.IMPORTANCE Ferrets have been widely used to study pathogenesis, immunity and transmission following human influenza virus infections, however far less is known regarding the utility of the ferret model to study hRSV infections. Following intranasal (IN) infection of adult ferrets with the well characterised Long or A2 strains of hRSV, we report virus replication and cytokine induction in the upper and lower airways, as well as the development of virus-specific humoral responses. Importantly, we demonstrate transmission of hRSV from experimentally infected donor ferrets to co-housed naïve recipients. Together, these findings significantly enhance our understanding of the utility of the ferret as a small animal model to investigate aspects of hRSV pathogenesis and immunity

The effect of entomopathogenic fungal culture filtrate on the immune response of the greater wax moth, Galleria mellonella

Galleria mellonella is a well-established model species regularly employed in the study of the insect immune response at cellular and humoral levels to investigate fungal pathogenesis and biocontrol agents. A cellular and proteomic analysis of the effect of culture filtrate of three entomopathogenic fungi (EPF) species on the immune system of G. mellonella was performed. Treatment with Beauveria caledonica and Metarhizium anisopliae 96 h culture filtrate facilitated a significantly increased yeast cell density in larvae (3-fold and 3.8-fold, respectively). Larvae co-injected with either M. anisopliae or B. caledonica culture filtrate and Candida albicans showed significantly increased mortality. The same was not seen for larvae injected with Beauveria bassiana filtrate. Together these results suggest that B. caledonica and M. anisopliae filtrate are modulating the insect immune system allowing a subsequent pathogen to proliferate. B. caledonica and M. anisopliae culture filtrates impact upon the larval prophenoloxidase (ProPO) cascade (e.g. ProPO activating factor 3 and proPO activating enzyme 3 were increased in abundance relative to controls), while B. bassiana treated larvae displayed higher abundances of alpha-esterase when compared to control larvae (2.4-fold greater) and larvae treated with M. anisopliae and B. caledonica. Treatment with EPF culture filtrate had a significant effect on antimicrobial peptide abundances particularly in M. anisopliae treated larvae where cecropin-D precursor, hemolin and gloverin were differentially abundant in comparison to controls. Differences in proteomic profiles for different treatments may reflect or even partially explain the differences in their immunomodulatory potential. Screening EPF for their ability to modulate the insect immune response represents a means of assessing EPF for use as biocontrol agents, particularly if the goal is to use them in combination with other control agents. Additionally EPF represent a valuable resource pool in our search for natural products with insect immunomodulatory and biocontrol properties

Interval between infections and viral hierarchy are determinants of viral interference following influenza virus infection in a ferret model

Background.Epidemiological studies suggest that, following infection with influenza virus, there is a short period during which a host experiences a lower susceptibility to infection with other influenza viruses. This viral interference appears to be independent of any antigenic similarities between the viruses. We used the ferret model of human influenza to systematically investigate viral interference. Methods.Ferrets were first infected then challenged 1-14 days later with pairs of influenza A(H1N1)pdm09, influenza A(H3N2), and influenza B viruses circulating in 2009 and 2010. Results.Viral interference was observed when the interval between initiation of primary infection and subsequent challenge was <1 week. This effect was virus specific and occurred between antigenically related and unrelated viruses. Coinfections occurred when 1 or 3 days separated infections. Ongoing shedding from the primary virus infection was associated with viral interference after the secondary challenge. Conclusions.The interval between infections and the sequential combination of viruses were important determinants of viral interference. The influenza viruses in this study appear to have an ordered hierarchy according to their ability to block or delay infection, which may contribute to the dominance of different viruses often seen in an influenza season

Re-exposure behaviour of Model R1 for different IFN production rates.

<p>A smaller IFN production for the primary virus for Model R1 does not lead to any qualitative difference, in terms of the dependence of model behaviours for the challenge virus on the IEI, from the case of very large IFN production rate of the first virus. The pattern is also independent of the choice of <i>q</i>₂. The meaning of each colour is explained in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g008" target="_blank">Fig 8</a>. They all exhibit four types of behaviours (seen vertically, separated by dashed lines) and within each type the phase decomposition and their order are preserved.</p

Innate Immunity and the Inter-exposure Interval Determine the Dynamics of Secondary Influenza Virus Infection and Explain Observed Viral Hierarchies

<div><p>Influenza is an infectious disease that primarily attacks the respiratory system. Innate immunity provides both a very early defense to influenza virus invasion and an effective control of viral growth. Previous modelling studies of virus–innate immune response interactions have focused on infection with a single virus and, while improving our understanding of viral and immune dynamics, have been unable to effectively evaluate the relative feasibility of different hypothesised mechanisms of antiviral immunity. In recent experiments, we have applied consecutive exposures to different virus strains in a ferret model, and demonstrated that viruses differed in their ability to induce a state of temporary immunity or viral interference capable of modifying the infection kinetics of the subsequent exposure. These results imply that virus-induced early immune responses may be responsible for the observed viral hierarchy. Here we introduce and analyse a family of within-host models of re-infection viral kinetics which allow for different viruses to stimulate the innate immune response to different degrees. The proposed models differ in their hypothesised mechanisms of action of the non-specific innate immune response. We compare these alternative models in terms of their abilities to reproduce the re-exposure data. Our results show that 1) a model with viral control mediated solely by a virus-resistant state, as commonly considered in the literature, is not able to reproduce the observed viral hierarchy; 2) the synchronised and desynchronised behaviour of consecutive virus infections is highly dependent upon the interval between primary virus and challenge virus exposures and is consistent with virus-dependent stimulation of the innate immune response. Our study provides the first mechanistic explanation for the recently observed influenza viral hierarchies and demonstrates the importance of understanding the host response to multi-strain viral infections. Re-exposure experiments provide a new paradigm in which to study the immune response to influenza and its role in viral control.</p></div

Re-exposure experimental data showing the four dominant patterns observed in the viral kinetics for primary–challenge virus pairs.

<p>The data shown here are distinct from those shown in Figs <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g001" target="_blank">1</a> and <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g002" target="_blank">2</a>, where the same phenomena may also be observed, and a subset of the full data presented in [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref010" target="_blank">10</a>]. Top panels show the case of co-infection, whereby both the primary (H1N1) and challenge (H3N2) viruses experience a synchronised increase in the very early stage of infection, followed by a synchronised decrease. Panels in the second row show examples of delayed infection, in which an initial synchronised decrease gives way to growth and successful infection with the challenge virus. The undetectable points between days 15 and 19 for the challenge virus in the right figure (second row) show a rapid decrease to undetectable viral level followed by a rapid upstroke back to a detectable level. Desynchronised viral kinetics in the early stage of infection are also observed for short IEIs, with examples shown in the third row of panels. The last well-observed pattern is that of a complete block, whereby the challenge virus is unable to replicate to a productive infection level (bottom panel). All symbols are the same as those in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.g001" target="_blank">Fig 1</a>. Data used from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004334#pcbi.1004334.ref010" target="_blank">10</a>].</p