Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and “synthetic” SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV

IFN-Lambda (IFN-λ) Is Expressed in a Tissue-Dependent Fashion and Primarily Acts on Epithelial Cells In Vivo

Interferons (IFN) exert antiviral, immunomodulatory and cytostatic activities. IFN-α/β (type I IFN) and IFN-λ (type III IFN) bind distinct receptors, but regulate similar sets of genes and exhibit strikingly similar biological activities. We analyzed to what extent the IFN-α/β and IFN-λ systems overlap in vivo in terms of expression and response. We observed a certain degree of tissue specificity in the production of IFN-λ. In the brain, IFN-α/β was readily produced after infection with various RNA viruses, whereas expression of IFN-λ was low in this organ. In the liver, virus infection induced the expression of both IFN-α/β and IFN-λ genes. Plasmid electrotransfer-mediated in vivo expression of individual IFN genes allowed the tissue and cell specificities of the responses to systemic IFN-α/β and IFN-λ to be compared. The response to IFN-λ correlated with expression of the α subunit of the IFN-λ receptor (IL-28Rα). The IFN-λ response was prominent in the stomach, intestine and lungs, but very low in the central nervous system and spleen. At the cellular level, the response to IFN-λ in kidney and brain was restricted to epithelial cells. In contrast, the response to IFN-α/β was observed in various cell types in these organs, and was most prominent in endothelial cells. Thus, the IFN-λ system probably evolved to specifically protect epithelia. IFN-λ might contribute to the prevention of viral invasion through skin and mucosal surfaces

Bunyaviridae RNA Polymerases (L-Protein) Have an N-Terminal, Influenza-Like Endonuclease Domain, Essential for Viral Cap-Dependent Transcription

Bunyaviruses are a large family of segmented RNA viruses which, like influenza virus, use a cap-snatching mechanism for transcription whereby short capped primers derived by endonucleolytic cleavage of host mRNAs are used by the viral RNA-dependent RNA polymerase (L-protein) to transcribe viral mRNAs. It was recently shown that the cap-snatching endonuclease of influenza virus resides in a discrete N-terminal domain of the PA polymerase subunit. Here we structurally and functionally characterize a similar endonuclease in La Crosse orthobunyavirus (LACV) L-protein. We expressed N-terminal fragments of the LACV L-protein and found that residues 1-180 have metal binding and divalent cation dependent nuclease activity analogous to that of influenza virus endonuclease. The 2.2 Å resolution X-ray crystal structure of the domain confirms that LACV and influenza endonucleases have similar overall folds and identical two metal binding active sites. The in vitro activity of the LACV endonuclease could be abolished by point mutations in the active site or by binding 2,4-dioxo-4-phenylbutanoic acid (DPBA), a known influenza virus endonuclease inhibitor. A crystal structure with bound DPBA shows the inhibitor chelating two active site manganese ions. The essential role of this endonuclease in cap-dependent transcription was demonstrated by the loss of transcriptional activity in a RNP reconstitution system in cells upon making the same point mutations in the context of the full-length LACV L-protein. Using structure based sequence alignments we show that a similar endonuclease almost certainly exists at the N-terminus of L-proteins or PA polymerase subunits of essentially all known negative strand and cap-snatching segmented RNA viruses including arenaviruses (2 segments), bunyaviruses (3 segments), tenuiviruses (4–6 segments), and orthomyxoviruses (6–8 segments). This correspondence, together with the well-known mapping of the conserved polymerase motifs to the central regions of the L-protein and influenza PB1 subunit, suggests that L-proteins might be architecturally, and functionally equivalent to a concatemer of the three orthomyxovirus polymerase subunits in the order PA-PB1-PB2. Furthermore, our structure of a known influenza endonuclease inhibitor bound to LACV endonuclease suggests that compounds targeting a potentially broad spectrum of segmented RNA viruses, several of which are serious or emerging human, animal and plant pathogens, could be developed using structure-based optimisation

Processing of Genome 5′ Termini as a Strategy of Negative-Strand RNA Viruses to Avoid RIG-I-Dependent Interferon Induction

Innate immunity is critically dependent on the rapid production of interferon in response to intruding viruses. The intracellular pathogen recognition receptors RIG-I and MDA5 are essential for interferon induction by viral RNAs containing 5′ triphosphates or double-stranded structures, respectively. Viruses with a negative-stranded RNA genome are an important group of pathogens causing emerging and re-emerging diseases. We investigated the ability of genomic RNAs from substantial representatives of this virus group to induce interferon via RIG-I or MDA5. RNAs isolated from particles of Ebola virus, Nipah virus, Lassa virus, and Rift Valley fever virus strongly activated the interferon-beta promoter. Knockdown experiments demonstrated that interferon induction depended on RIG-I, but not MDA5, and phosphatase treatment revealed a requirement for the RNA 5′ triphosphate group. In contrast, genomic RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus did not trigger interferon induction. Sensitivity of these RNAs to a 5′ monophosphate-specific exonuclease indicates that the RIG-I-activating 5′ triphosphate group was removed post-transcriptionally by a viral function. Consequently, RIG-I is unable to bind the RNAs of Hantaan virus, Crimean-Congo hemorrhagic fever virus and Borna disease virus. These results establish RIG-I as a major intracellular recognition receptor for the genome of most negative-strand RNA viruses and define the cleavage of triphosphates at the RNA 5′ end as a strategy of viruses to evade the innate immune response

Completion of Hepatitis C Virus Replication Cycle in Heterokaryons Excludes Dominant Restrictions in Human Non-liver and Mouse Liver Cell Lines

Hepatitis C virus (HCV) is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti

p53 Activation following Rift Valley Fever Virus Infection Contributes to Cell Death and Viral Production

Rift Valley fever virus (RVFV) is an emerging viral zoonosis that is responsible for devastating outbreaks among livestock and is capable of causing potentially fatal disease in humans. Studies have shown that upon infection, certain viruses have the capability of utilizing particular cellular signaling pathways to propagate viral infection. Activation of p53 is important for the DNA damage signaling cascade, initiation of apoptosis, cell cycle arrest and transcriptional regulation of multiple genes. The current study focuses on the role of p53 signaling in RVFV infection and viral replication. These results show an up-regulation of p53 phosphorylation at several serine sites after RVFV MP-12 infection that is highly dependent on the viral protein NSs. qRT-PCR data showed a transcriptional up-regulation of several p53 targeted genes involved in cell cycle and apoptosis regulation following RVFV infection. Cell viability assays demonstrate that loss of p53 results in less RVFV induced cell death. Furthermore, decreased viral titers in p53 null cells indicate that RVFV utilizes p53 to enhance viral production. Collectively, these experiments indicate that the p53 signaling pathway is utilized during RVFV infection to induce cell death and increase viral production

Immunological Mechanisms Mediating Hantavirus Persistence in Rodent Reservoirs

Hantaviruses, similar to several emerging zoonotic viruses, persistently infect their natural reservoir hosts, without causing overt signs of disease. Spillover to incidental human hosts results in morbidity and mortality mediated by excessive proinflammatory and cellular immune responses. The mechanisms mediating the persistence of hantaviruses and the absence of clinical symptoms in rodent reservoirs are only starting to be uncovered. Recent studies indicate that during hantavirus infection, proinflammatory and antiviral responses are reduced and regulatory responses are elevated at sites of increased virus replication in rodents. The recent discovery of structural and non-structural proteins that suppress type I interferon responses in humans suggests that immune responses in rodent hosts could be mediated directly by the virus. Alternatively, several host factors, including sex steroids, glucocorticoids, and genetic factors, are reported to alter host susceptibility and may contribute to persistence of hantaviruses in rodents. Humans and reservoir hosts differ in infection outcomes and in immune responses to hantavirus infection; thus, understanding the mechanisms mediating viral persistence and the absence of disease in rodents may provide insight into the prevention and treatment of disease in humans. Consideration of the coevolutionary mechanisms mediating hantaviral persistence and rodent host survival is providing insight into the mechanisms by which zoonotic viruses have remained in the environment for millions of years and continue to be transmitted to humans