Journal of General Virology - Current Issue

Infectious bursal disease virus (IBDV) causes an endemic immunosuppressive disease in chickens. Prior exposure to IBDV influences the pathogenesis and shedding of chicken strains of influenza A virus (IAV), but its effect on waterfowl strains is poorly understood. To address this, we inoculated 14-day-old specific pathogen-free chickens with low pathogenicity avian influenza strain A/Mallard/Alberta/156/01 (H3N8) and compared the replication, shedding, pathogenesis, transmission and intra-host evolution between immunocompetent chickens and chickens that had IBDV-mediated immune dysregulation due to a prior infection with strain F52/70 at 2 days of age. The IAV replicated in the upper respiratory tract, and the virus was shed from the oropharyngeal cavity, but there was no shedding from the cloaca and no transmission to sentinel chickens. IAV replication in chickens was associated with amino acid substitutions in the polymerase complex and HA. Prior IBDV infection had no significant effect on IAV pathogenicity, replication or shedding and had a modest effect on IAV diversity, increasing the number of amino acid substitutions from an average of 2.50 substitutions per sample (sd±1.83) in the Mock/IAV group to 4.75 (sd±1.81) in the IBDV/IAV group (P<0.01). Taken together, our data suggest that IBDV is unlikely to play a major role in the spillover or spread of waterfowl IAV strains in chicken flocks, although it could expand IAV diversity. This information is useful for informing preventative measures for controlling IAV in poultry flocks.

While rapid death is the usual outcome of high pathogenicity avian influenza virus (HPAIV) infection in gallinaceous poultry, HPAIV-infected ducks usually present milder clinical signs and shed virus for a prolonged time. The difference in disease severity has been linked to a more rapid type I IFN immune response and reduced proinflammatory cytokine expression in ducks compared to chickens. To investigate the role of the early antiviral innate immune response in controlling viral replication in ducks, we evaluated the effects of ruxolitinib, a Janus kinase–signal transducer and activator of transcription (JAK–STAT) pathway inhibitor known to dampen IFN signalling in mammals. We first optimized a treatment protocol in 2-week-old ducklings and showed that repeated intracoelomic injections of ruxolitinib significantly decreased IFN-stimulated gene (ISG) mRNA levels in the lung, while oral administration was ineffective. In subsequent infection experiments with an H5N9 HPAIV strain, ruxolitinib treatment led to an earlier peak of viral shedding and increased viral RNA levels in respiratory tissues, which however was not associated with increased expression of clinical signs. Analysis of host immune gene expression in the respiratory tract confirmed a transient suppression of ISG expression coincident with ruxolitinib treatment, followed by a recovery once treatment was ceased. This work provides the first demonstration of the effectiveness of ruxolitinib at inhibiting the antiviral innate immune response in birds, causing increased levels of virus replication when administered to HPAIV-infected ducks.

Processing bodies (P-bodies/PBs) are non-membranous foci involved in coordinating RNA fate by regulating translation and mRNA decay. In this study, we characterize the anti-viral factor Shiftless (SHFL) as a potent disruptor of PB dynamics. We show SHFL expression restricts PB accumulation even in the context of oxidative stress, suggesting that SHFL expression impedes PB formation. Mutational approaches revealed that SHFL RNA-binding activity is not required to restrict PB formation. However, we have identified a new region of SHFL, a bridge between two distant SHFL domains, as necessary for SHFL-mediated PB disruption. Furthermore, we show that SHFL’s ability to disrupt PB formation also impacts its anti-viral activity during infection by the gammaherpesvirus Kaposi’s sarcoma-associated herpesvirus (KSHV). While WT SHFL efficiently restricts KSHV lytic reactivation, SHFL mutants defective in PB disruption no longer restrict KSHV reactivation. SHFL-mediated PB disruption also leads to increased expression of several anti-viral cytokines, further emphasizing the connection among SHFL, PB dynamics and the SHFL-dependent anti-viral state. Taken together, our observations suggest a role of SHFL in inhibiting PB formation to restrict KSHV lytic replication, reinforcing the importance of crosstalk between RNA fate and the innate immune response to viral infection.

The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small-animal model for congenital cytomegalovirus, a leading cause of cognitive impairment and hearing loss in newborns. GPCMV encodes human cytomegalovirus (HCMV) homologues of viral entry glycoprotein complexes, which are neutralizing-antibody vaccine targets. As with HCMV, GPCMV has two pathways of cell entry (direct and endocytic). Specific viral gH/gL-based complexes are necessary for receptor interaction and cell entry: gH/gL/gO trimer (direct) and pentamer complex (PC) (endocytic). Both pathways also require gB as the fusogenic protein. Direct GPCMV cell entry requires platelet-derived growth factor receptor alpha (PDGFRA), but an endocytic PC receptor remains unknown. We hypothesized that cellular knockout of direct and endocytic receptors would completely block infection, which cannot be achieved by gB-based antibodies. Candidate receptors including neuropilin proteins (NRP1, NRP2) and CD147 present on all established guinea pig cell lines were selected based on importance as common virus receptors or in fetal development. Results demonstrated that NRP2 interacted with PC, unlike NRP1 or CD147, in immunoprecipitation assays and eliminated NRP1/NRP2 heterodimer receptor interaction. The viral trimer only interacted with PDGFRA. Double knockout of PDGFRA/NRP2 completely blocked GPCMV infection. In contrast, the CD147/PDGFRA double knockout had limited GPCMV inhibition, and the single knockout of CD147 had no impact. Knockout of the various receptors had no effect on control HSV-1 infection. Ectopic expression of guinea pig cell receptors restored GPCMV infection but not human NRP2/PDGFRA, indicating a basis for the species-specific barrier for GPCMV and HCMV infection. Overall, results increase the translational relevance of GPCMV for the development of CMV intervention strategies.

Herpes simplex virus (HSV)-1 infection of cortical neurones is a leading cause of encephalitis. Whilst we have substantial knowledge about the molecular virology of HSV-1 lytic infection in cells of the periphery, like keratinocytes or fibroblasts, we know much less about infection of human neurones owing to the challenges of working with neuronal cell-based models. Here, we demonstrate the use of a human induced pluripotent stem cell-derived cortical neurone model (i3Neurones) for HSV-1 infection. i3Neurones are highly scalable and can be rapidly and efficiently differentiated into an isogenic population of cortical glutamatergic neurones. We show that i3Neurones support the full HSV-1 lytic replication cycle. We present an optimized protocol for the infection of i3Neurones with HSV-1 that allows their synchronous infection at near-100% efficiency and optimized fixation methods that preserve organelle and neurite structure for immunocytochemistry analysis. Our study highlights i3Neurones as a robust, scalable platform for microscopy and biochemical studies of HSV-1 and other neurotropic pathogens.