Russell K. Durbin

Differential Type I Interferon Induction by Respiratory Syncytial Virus and Influenza A Virus In Vivo

ABSTRACT Type I interferon (IFN) induction is an immediate response to virus infection, and very high levels of these cytokines are produced when the Toll-like receptors (TLRs) expressed at high levels by plasmacytoid dendritic cells (pDCs) are triggered by viral nucleic acids. Unlike many RNA viruses, respiratory syncytial virus (RSV) does not appear to activate pDCs through their TLRs and it is not clear how this difference affects IFN-α/β induction in vivo. In this study, we investigated type I IFN production triggered by RSV or influenza A virus infection of BALB/c mice and found that while both viruses induced IFN-α/β production by pDCs in vitro, only influenza virus infection could stimulate type I IFN synthesis by pDCs in vivo. In situ hybridization studies demonstrated that the infected respiratory epithelium was a major source of IFN-α/β in response to either infection, but in pDC-depleted animals only type I IFN induction by influenza virus was impaired.

Lambda Interferon Is the Predominant Interferon Induced by Influenza A Virus Infection In Vivo

ABSTRACT The type I alpha/beta interferons (IFN-α/β) are known to play an important role in host defense against influenza A virus infection, but we have now discovered that the recently identified type III IFNs (IFN-λ) constitute the major response to intranasal infection with this virus. Type III IFNs were present at much higher levels than type I IFNs in the lungs of infected mice, and the enhanced susceptibility of STAT2−/− animals demonstrated that only signaling through the IFN-α/β or IFN-λ pathways was sufficient to mediate protection. This finding offers a possible explanation for the similar levels of antiviral protection found in wild-type (WT) mice and in animals lacking a functional type I IFN receptor (IFNAR−/−) but also argues that our current understanding of type III IFN induction is incomplete. While murine IFN-λ production is thought to depend on signaling through the type I IFN receptor, we demonstrate that intranasal influenza A virus infection leads to the robust type III IFN induction in the lungs of both WT and IFNAR−/− mice. This is consistent with previous studies showing that IFNAR-mediated protection is redundant for mucosal influenza virus infection and with data showing that the type III IFN receptor is expressed primarily by epithelial cells. However, the overlapping effects of these two cytokine families are limited by their differential receptor expression, with a requirement for IFN-α/β signaling in combating systemic disease.

Association of the sindbis virus RNA methyltransferase activity with the nonstructural protein nsP1

SVLM21 is a mutant of Sindbis virus, which in contrast to SVSTD, is able to replicate in Aedes albopictus mosquito cells deprived of methionine. We have obtained evidence that the basis of this low methionine-resistance (LMR) phenotype is the generation of an altered RNA methyltransferase with an increased affinity for S-adenosylmethionine (ado met). We now report that following the substitution of the nucleotide sequence, 126-504, from SVLM21 cDNA for the corresponding sequence of the Toto 1101 plasmid (infectious Sindbis viral RNA can be transcribed from this plasmid) we were able to generate recombinant Sindbis virus (SVMS-65a) with the LMR phenotype. (SVTOTO virus derived from Toto 1101, like SVSTD, lacks the LMR phenotype.) As was the case with SVLM21, SVMS-65a not only possessed the LMR phenotype but also showed an increased sensitivity to Neplanocin A, a potent inhibitor of S-adenosylhomocysteine (ado hcy) hydrolase. Sequencing of the nucleotide 126-504 region from SVLM21 revealed two mutations; these mutations occurred in adjacent codons and lead to two predicted amino acid changes in the SV nsPl protein; at residue 87, from Arg to Leu, and at residue 88 from Ser to Cys. Since the nucleotide sequence 126-504 lies entirely within the gene for nsP1, we conclude that the RNA methyltransferase activity generated by SV is associated with nsP1. We suggest that residues 87 and 88 in nsP1, where the amino acid changes in SVLM21 nsP1 have occurred, are at or near the binding site for ado met; we also suggest that these changes in nsP1 are responsible for the increased affinity of the SVLM21 RNA methyltransferase for ado met and thereby for the LMR phenotype. Alternatively, it is possible that the binding site for ado met is elsewhere on nsP1 or even on another protein, and that the changes at residues 87 and 88 lead to an alteration of the binding site.

Protection against Respiratory Syncytial Virus by a Recombinant Newcastle Disease Virus Vector

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infants and the elderly, but no safe and effective RSV vaccine is yet available. For reasons that are not well understood, RSV is only weakly immunogenic, and reinfection occurs throughout life. This has complicated the search for an effective live attenuated viral vaccine, and past trials with inactivated virus preparations have led to enhanced immunopathology following natural infection. We have tested the hypothesis that weak stimulation of innate immunity by RSV correlates with ineffective adaptive responses by asking whether expression of the fusion glycoprotein of RSV by Newcastle disease virus (NDV) would stimulate a more robust immune response to RSV than primary RSV infection. NDV is a potent inducer of both alpha/beta interferon (IFN-α/β) production and dendritic cell maturation, while RSV is not. When a recombinant NDV expressing the RSV fusion glycoprotein was administered to BALB/c mice, they were protected from RSV challenge, and this protection correlated with a robust anti-F CD8+ T-cell response. The effectiveness of this vaccine construct reflects the differential abilities of NDV and RSV to promote dendritic cell maturation and is retained even in the absence of a functional IFN-α/β receptor.

Sindbis virus mutants resistant to mycophenolic acid and ribavirin

Previous work from this laboratory has demonstrated a correlation between the inhibition by ribavirin (Rbv), mycophenolic acid (MPA), or 2-amino thiadiazole (TDA) of Sindbis virus replication in Aedes albopictus mosquito cells and a reduction in cellular GTP levels. This reduction in GTP results from the inhibition by these drugs of inosine monophosphate dehydrogenase (IMPDH), the first enzyme specific for the de novo synthesis of GMP. By serial passage of SV in A. albopictus cells in the presence of 25 microM MPA, we have now isolated viral mutants which are highly resistant not only to MPA but also to Rbv and TDA. For example, whereas 500 microM Rbv reduced the plaquing efficiency of SVSTD by at least 10(6)-fold, the same concentration of Rbv reduced the plaquing efficiency of the MPA-resistant mutants less than 5-fold. This is the first example of a viral mutant resistant to the antiviral compound Rbv.

Role for Innate IFNs in Determining Respiratory Syncytial Virus Immunopathology

Respiratory syncytial virus (RSV) is the major cause of severe lower airway disease in infants and young children, but no safe and effective RSV vaccine is yet available. The difficulties involved in RSV vaccine development were recognized in an early vaccine trial, when children immunized with a formalin-inactivated virus preparation experienced enhanced illness after natural infection. Subsequent research in animal models has shown that the vaccine-enhanced disease is mediated at least in part by memory cells producing Th2 cytokines. Previously we had observed enhanced, eosinophilic lung pathology during primary infection of IFN-deficient STAT1−/− mice that are incapable of generating Th1 CD4+ cells. To determine whether these effects depended only on Th2 cytokine secretion or involved other aspects of IFN signaling, we infected a series of 129SvEv knockout mice lacking the IFN-αβR (IFN-αβR−/−), the IFN-γR (IFN-γR−/−), or both receptors (IFN-αβγR−/−). Although both the IFN-γR−/− and the IFN-αβγR−/− animals generated strong Th2 responses to RSV-F protein epitopes, predominantly eosinophilic lung disease was limited to mice lacking both IFNRs. Although the absolute numbers of eosinophils in BAL fluids were similar between the strains, very few CD8+ T cells could be detected in lungs of IFN-αβγR−/− animals, leaving eosinophils as the predominant leukocyte. Thus, although CD4+ Th2 cell differentiation is necessary for the development of allergic-type inflammation after infection and appears to be unaffected by type I IFNs, innate IFNs clearly have an important role in determining the nature and severity of RSV disease.

PKR Protection Against Intranasal Vesicular Stomatitis Virus Infection Is Mouse Strain Dependent

The interferon-induced antiviral state is mediated by interferon-stimulated genes that are upregulated in concert after stimulation by type I interferons. Because so many viruses encode strategies to inactivate the interferon-inducible double-stranded RNA (dsRNA)-dependent protein kinase PKR, this protein is likely to be a major player in antiviral defense. Here we demonstrate the increased susceptibility of PKR-/- animals to vesicular stomatitis virus (VSV) by the intranasal route, but also demonstrate that the protective effects of PKR are mouse strain dependent. We have found the difference between wild-type-BALB/c and 129SvEv animals to be on the order of 5 logs, with high levels of virus present in the lungs of BALB/c but not 129SvEv animals. To evaluate the sensitivity of PKR-/- mice to VSV clearly, the PKR mutation was bred onto the resistant 129SvEv background. The increased sensitivity of PKR-/- mice, compared to PKR+/+ strain-matched controls, is on the order of 10-fold as measured by median lethal dose (LD50). PKR-/- 129 mice support VSV replication in the lung unlike controls. While this result clearly demonstrates an important role for PKR in protection against VSV infection of the lung, it also underlines the importance of other host factors in containing a viral infection.

A mutant of sindbis virus with a host-dependent defect in maturation associated with hyperglycosylation of E2.

Following serial passage of Sindbis virus (SV) on Aedes albopictus mosquito cells a mutant (SVap15/21) was isolated which in chick cells produced small plaques and was temperature sensitive (ts). At 34.5 degrees this mutant replicated normally in mosquito cells, but only poorly in chick or BHK cells. In the vertebrate cells SVap15/21 was RNA+ at both 34.5 and 40 degrees and on the basis of complementation tests carried out at 40 degrees, was assigned to complementation group E. The block in the replication of this mutant, like that of ts20, the prototype mutant of complementation group E, was at the level of nucleocapsid envelopment. The PE2 and E2 glycoproteins of SVap15/21 were found to be hyperglycosylated relative to the corresponding glycoproteins of the parent virus (SVstd). Analysis of revertants of SVap15/21 suggests a causal relationship between PE2 and E2 hyperglycosylation and the host-specific defect in virus maturation. The association of a host-specific defect in virion assembly with hyperglycosylation of a viral structural protein points to the potential importance of host-specific glycosylation patterns in the determination of viral host range.

Chinchilla and Murine Models of Upper Respiratory Tract Infections with Respiratory Syncytial Virus

ABSTRACT Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in infants and the elderly. While the primary infection is the most serious, reinfection of the upper airway throughout life is the rule. Although relatively little is known about either RSV infection of the upper respiratory tract or host mucosal immunity to RSV, recent literature suggests that RSV is the predominant viral pathogen predisposing to bacterial otitis media (OM). Herein, we describe mouse and chinchilla models of RSV infection of the nasopharynx and Eustachian tube. Both rodent hosts were susceptible to RSV infection of the upper airway following intranasal challenge; however, the chinchilla proved to be more permissive than the mouse. The chinchilla model will likely be extremely useful to test the role of RSV in bacterial OM and the efficacy of RSV vaccine candidates designed to provide mucosal and cytotoxic T-lymphocyte immunity. Ultimately, we hope to investigate the relative ability of these candidates to potentially protect against viral predisposal to bacterial OM.

Signaling through the Prostaglandin I2 Receptor IP Protects against Respiratory Syncytial Virus-Induced Illness

ABSTRACT The role of prostanoids in modulating respiratory syncytial virus (RSV) infection is unknown. We found that RSV infection in mice increases production of prostaglandin I2 (PGI2). Mice that overexpress PGI2 synthase selectively in bronchial epithelium are protected against RSV-induced weight loss and have decreased peak viral replication and gamma interferon levels in the lung compared to nontransgenic littermates. In contrast, mice deficient in the PGI2 receptor IP have exacerbated RSV-induced weight loss with delayed viral clearance and increased levels of gamma interferon in the lung compared to wild-type mice. These results suggest that signaling through IP has antiviral effects while protecting against RSV-induced illness and that PGI2 is a potential therapeutic target in the treatment of RSV.