Christopher P Malinoski’s research while affiliated with University of Connecticut and other places

This dissertation explores the complex nature of the influenza virus quasispecies. Influenza populations exist as a mixture of mostly replication-deficient biologically-active particles. These particles are measured and quantified based upon the phenotype they manifest upon entry into the host. Three subpopulations are characterized: infectious, interferon-inducing, and interferon induction-suppressing particles. It has been observed that only 5 out of every 100 particles of influenza are infectious. A novel biological assay was developed and, for the first time, directly demonstrated that infectious particles are the progenitors of all other virus particles. From the disparity observed to exist between the numbers of physical and infectious particles, a mathematical approach is presented which quantifies the frequency of expression of individual gene segments. Interferon-inducing particles are demonstrated to be resistant to physical and chemical inactivation when measured in avian cells –the best evidence to date that the interferon-inducing moiety is preformed within the virion. In contrast, interferon-inducing particles are inactivated at a rate consistent with inactivation of the NS gene in mammalian cells, suggesting that primary transcription is necessary for interferon-induction in these cells. Interferon induction-suppressing particles inhibit interferon production in cells otherwise programed to induce. This activity is nonspecific, blocking interferon-induction by viruses belonging to multiple families, and is dependent upon expression of the NS1 protein. A mathematical model, based on a random (Poisson) distribution of virus particles amongst the cell monolayer, is presented that predicts the fraction of cells that will induce interferon, dependent upon the ratio of interferon-inducing and interferon induction-suppressing particles present in the virus stock. A novel method for quantifying interferon-inducing particles in preparations with high interferon induction-suppressing particle content is described. A highly potent interferon induction-suppression activity was also found to be associated with exposure to lipopolysaccharide. The functional heterogeneity demonstrated by a single strain of influenza was quantified, utilizing interferon-induction and interferon induction-suppression as phenotypic markers of the quasispecies. The activities of 117 plaque-derived isolates were measured, and variability in these phenotypes was observed over a 1000-fold range. The genetic basis of this phenotypic variance was investigated through sequencing the NS genes of several isolates demonstrating extreme phenotypes.

Influenza virus populations contain several subpopulations of noninfectious biologically active particles that are measured by the unique phenotypes they express. Two of these subpopulations were studied: (1) interferon (IFN)-inducing particles (IFP) and (2) IFN induction-suppressing particles (ISP). ISP are dominant in cells coinfected with one or more IFP; they completely suppress IFN production in cells otherwise programmed to induce it. Influenza virus ISP were shown to act in host cells in a nonspecific and global manner, suppressing IFN induction independent of the family of viruses serving as IFN inducers. ISP must be present within the first 3 h of coinfection with IFP to be maximally effective; by 7 hpi IFN induction/production is refractory to the action of superinfecting ISP. UV target and thermal inactivation analyses revealed that ISP activity was dependent solely on the expression of the NS gene. Low doses of UV radiation enhanced by ∼10-fold the already high IFN-inducing capacity of a virus that expressed truncated NS1. There was no change in the number of IFP, implying that the production of IFN/cell had increased. We postulated that preventing degradation of cellular RNA pol II by viral polymerase prolonged the transcription of cellular mRNA, including IFN mRNA, to enhance the IFN-inducing capacity of the cell without any increase in the number of IFP. These studies point to the dueling roles of IFP and ISP in modulating IFN induction/production, the former activity being critical to the efficacy of live attenuated influenza vaccines.

The interferon (IFN)-inducing capacity of influenza virus plays a significant role in the efficacy of candidate live attenuated influenza vaccines (LAIVs). IFN is induced by a subpopulation of noninfectious biologically active particles (niBAPs) that can be defined and quantified as IFN-inducing particles (IFPs). When chicken embryonic cells were infected with increasing multiplicities of IFP (m(ifp)), the amount of IFN produced was that expected from a Poisson distribution of cells infected with ≥1 IFP. Problematically, some isolates of influenza virus induced less IFN than expected at higher m(ifp). We postulated that these stocks contained another subpopulation of niBAP, IFN induction-suppressing particles (ISPs). A single ISP was assumed capable of preventing IFN production completely in cells coinfected with IFP. Virus stocks were reconstructed to contain a wide range of ratios of IFP:ISP and used to generate IFN-induction dose-response curves. The deviation of the observed yields of IFN from those expected if the virus stock consisted only of IFP fits well the results expected from a formulation of the Poisson distribution that provides the fraction of IFP-infected cells expected to become coinfected with ISP, and hence not yield IFN, as the ratio IFP:ISP decreases. The ideal LAIV might be thought to contain little or no ISP so as to maximize IFN production; however, the most effective LAIV appear to regulate the production of IFN. Thus, it is possible that an optimal ratio of IFP:ISP may exist to produce more effective LAIV, an event that may sometimes occur in nature, or be reconstructed.

During the course of codifying low pathogenicity avian influenza, viruses were tested for their capacity to induce type-I interferon (IFN) and to measure their content of IFN induction-suppressing particles (ISP). One isolate caused a >10-fold reduction in the yield of IFN from chicken embryonic cells co-infected with a virus that normally induces high yields of IFN. The apparent content of ISP was calculated to be approximately 100-fold higher than the number of physical particles of virus measured as hemagglutinating particles. This unrealistic interpretation prompted us to test for a soluble IFN induction-suppressing activity in the allantoic fluid freed of the virus by centrifugation. Indeed, the IFN induction-suppressing activity remained in the virus-free supernatant. The original virus stock subsequently was found to be contaminated with a Gram-negative bacterium, leading us to test lipopolysaccharide (LPS) as the putative IFN induction suppressor. Pure LPS mimicked in a similar dose-dependent manner the IFN induction-suppressing activity of the original allantoic fluid-derived virus, and the allantoic fluid freed of all virus and bacteria. The inhibition of viral-mediated type-I IFN induction by LPS was observed for viruses from 3 different families. These observations suggest that exposure of a host to endotoxin may compromise the IFN induction response of the innate immune system and thus exacerbate virus infection.