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1.  A Mouse Model for Betacoronavirus Subgroup 2c Using a Bat Coronavirus Strain HKU5 Variant 
mBio  2014;5(2):e00047-14.
ABSTRACT
Cross-species transmission of zoonotic coronaviruses (CoVs) can result in pandemic disease outbreaks. Middle East respiratory syndrome CoV (MERS-CoV), identified in 2012, has caused 182 cases to date, with ~43% mortality, and no small animal model has been reported. MERS-CoV and Pipistrellus bat coronavirus (BtCoV) strain HKU5 of Betacoronavirus (β-CoV) subgroup 2c share >65% identity at the amino acid level in several regions, including nonstructural protein 5 (nsp5) and the nucleocapsid (N) protein, which are significant drug and vaccine targets. BtCoV HKU5 has been described in silico but has not been shown to replicate in culture, thus hampering drug and vaccine studies against subgroup 2c β-CoVs. We report the synthetic reconstruction and testing of BtCoV HKU5 containing the severe acute respiratory syndrome (SARS)-CoV spike (S) glycoprotein ectodomain (BtCoV HKU5-SE). This virus replicates efficiently in cell culture and in young and aged mice, where the virus targets airway and alveolar epithelial cells. Unlike some subgroup 2b SARS-CoV vaccines that elicit a strong eosinophilia following challenge, we demonstrate that BtCoV HKU5 and MERS-CoV N-expressing Venezuelan equine encephalitis virus replicon particle (VRP) vaccines do not cause extensive eosinophilia following BtCoV HKU5-SE challenge. Passage of BtCoV HKU5-SE in young mice resulted in enhanced virulence, causing 20% weight loss, diffuse alveolar damage, and hyaline membrane formation in aged mice. Passaged virus was characterized by mutations in the nsp13, nsp14, open reading frame 5 (ORF5) and M genes. Finally, we identified an inhibitor active against the nsp5 proteases of subgroup 2c β-CoVs. Synthetic-genome platforms capable of reconstituting emerging zoonotic viral pathogens or their phylogenetic relatives provide new strategies for identifying broad-based therapeutics, evaluating vaccine outcomes, and studying viral pathogenesis.
IMPORTANCE
The 2012 outbreak of MERS-CoV raises the specter of another global epidemic, similar to the 2003 SARS-CoV epidemic. MERS-CoV is related to BtCoV HKU5 in target regions that are essential for drug and vaccine testing. Because no small animal model exists to evaluate MERS-CoV pathogenesis or to test vaccines, we constructed a recombinant BtCoV HKU5 that expressed a region of the SARS-CoV spike (S) glycoprotein, thereby allowing the recombinant virus to grow in cell culture and in mice. We show that this recombinant virus targets airway epithelial cells and causes disease in aged mice. We use this platform to (i) identify a broad-spectrum antiviral that can potentially inhibit viruses closely related to MERS-CoV, (ii) demonstrate the absence of increased eosinophilic immune pathology for MERS-CoV N protein-based vaccines, and (iii) mouse adapt this virus to identify viral genetic determinants of cross-species transmission and virulence. This study holds significance as a strategy to control newly emerging viruses.
doi:10.1128/mBio.00047-14
PMCID: PMC3977350  PMID: 24667706
2.  Regulatory B Cell (B10 Cell) Expansion During Listeria Infection Governs Innate and Cellular Immune Responses In Mice 
Pathogens use numerous methods to subvert host immune responses, including the modulation of host IL-10 production by diverse cell types. However, the B cell sources of IL-10 and their overall influence on innate and cellular immune responses have not been well characterized during infections. Using Listeria as a model pathogen, infection drove the acute expansion of a small subset of regulatory B cells (B10 cells) that potently suppress inflammation and autoimmunity through the production of IL-10. Unexpectedly, spleen bacteria loads were 92–97% lower in B10 cell-deficient CD19−/− mice, in mice depleted of mature B cells, and in mice treated with CD22 mAb to preferentially deplete B10 cells before infection. By contrast, the adoptive transfer of wild type B10 cells reduced bacterial clearance by 38-fold in CD19−/− mice through IL-10-dependent pathways. B10 cell depletion using CD22 mAb significantly enhanced macrophage phagocytosis of Listeria and their production of IFN-γ, TNF-α, and nitric oxide ex vivo. Accelerated bacteria clearance following B10 cell depletion significantly reduced Ag-specific CD4+ T cell proliferation and cytokine production, but did not alter CD8+ T cell responses. B10 cell regulatory function during innate immune responses was nonetheless dependent on cognate interactions with CD4+ T cells since B10 cells deficient in IL-10, MHC-II or IL-21 receptor expression did not influence Listeria clearance. Thus, Listeria manipulates immune responses through a strategy of immune evasion that involves the preferential expansion of endogenous B10 cells that regulate the magnitude and duration of both innate and cellular immune responses.
doi:10.4049/jimmunol.1201427
PMCID: PMC3552111  PMID: 23275601
B cells; Listeria monocytogenes; innate immunity; regulatory B cells; B10 cells
4.  Dendritic Cell Immunoreceptor Regulates Chikungunya Virus Pathogenesis in Mice 
Journal of Virology  2013;87(10):5697-5706.
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus responsible for recent epidemic outbreaks of debilitating disease in humans. Alphaviruses are known to interact with members of the C-type lectin receptor family of pattern recognition proteins, and given that the dendritic cell immunoreceptor (DCIR) is known to act as a negative regulator of the host inflammatory response and has previously been associated with rheumatoid arthritis, we evaluated DCIR's role in response to CHIKV infection. Although we observed an increase in the proportion of dendritic cells at the site of CHIKV infection at 24 to 36 h postinfection, these cells showed decreased cell surface DCIR, suggestive of DCIR triggering and internalization. In vitro, bone marrow-derived dendritic cells from DCIR-deficient (DCIR−/−) mice exhibited altered cytokine expression following exposure to CHIKV. DCIR−/− mice exhibited more severe disease signs than wild-type C57BL6/J mice following CHIKV infection, including a more rapid and more severe onset of virus-induced edema and enhanced weight loss. Histological examination revealed that DCIR-deficient animals exhibited increased inflammation and damage in both the fascia of the inoculated foot and the ankle joint, and DCIR deficiency skewed the CHIKV-induced cytokine response at the site of infection at multiple times postinfection. Early differences in virus-induced disease between C57BL6/J and DCIR−/− mice were independent of viral replication, while extended viral replication correlated with enhanced foot swelling and tissue inflammation and damage in DCIR−/− compared to C57BL6/J mice at 6 to 7 days postinfection. These results suggest that DCIR plays a protective role in limiting the CHIKV-induced inflammatory response and subsequent tissue and joint damage.
doi:10.1128/JVI.01611-12
PMCID: PMC3648201  PMID: 23487448
5.  The Viral Interferon Regulatory Factors of Kaposi's Sarcoma-Associated Herpesvirus Differ in Their Inhibition of Interferon Activation Mediated by Toll-Like Receptor 3 
Journal of Virology  2013;87(2):798-806.
Kaposi's sarcoma-associated herpesvirus (KSHV) infection is correlated with three human malignancies and can establish lifelong latent infection in multiple cell types within its human host. In order to establish and maintain infection, KSHV utilizes multiple mechanisms to evade the host immune response. One such mechanism is the expression of a family of genes with homology to cellular interferon (IFN) regulatory factors (IRFs), known as viral IRFs (vIRFs). We demonstrate here that KSHV vIRF1, -2, and -3 have a differential ability to block type I interferon signaling mediated by Toll-like receptor 3 (TLR3), a receptor we have previously shown to be activated upon KSHV infection. vIRF1, -2, and -3 inhibited TLR3-driven activation of IFN transcription reporters. However, only vIRF1 and vIRF2 inhibited increases in both IFN-β message and protein levels following TLR3 activation. The expression of vIRF1 and vIRF2 also allowed for increased replication of a virus known to activate TLR3 signaling. Furthermore, vIRF1 and vIRF2 may block TLR3-mediated signaling via different mechanisms. Altogether, this report indicates that vIRFs are able to block IFN mediated by TLRs but that each vIRF has a unique function and mechanism for blocking antiviral IFN responses.
doi:10.1128/JVI.01851-12
PMCID: PMC3554052  PMID: 23115281
6.  Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus 
PLoS Pathogens  2013;9(4):e1003312.
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.
Author Summary
Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes outbreaks of polyarthritis in humans, and is currently a threat to spread to the United States due to the presence of its mosquito vector, Aedes albopictus. At present, there is no licensed human vaccine or therapeutic available to protect against CHIKV infection. The primary goal of this study was to develop an antibody-based therapeutic agent against CHIKV. To do this, we developed a panel of 230 new mouse anti-CHIKV MAbs and tested them for their ability to neutralize infection of different CHIKV strains in cell culture. We identified 36 MAbs with broad neutralizing activity, and then tested several of these for their ability to protect immunocompromised Ifnar−/− mice against lethal CHIKV infection. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs limited the development of resistance and protected Ifnar−/− mice against disease even when given just 24 to 36 hours before CHIKV-induced death. Analogous protection against CHIKV-induced arthritis was seen in a disease model in wild type mice. Our data suggest that pairs of highly neutralizing MAbs may be a therapeutic option against CHIKV infection.
doi:10.1371/journal.ppat.1003312
PMCID: PMC3630103  PMID: 23637602
7.  Myd88-Dependent Toll-Like Receptor 7 Signaling Mediates Protection from Severe Ross River Virus-Induced Disease in Mice 
Journal of Virology  2012;86(19):10675-10685.
Arthralgia-associated alphaviruses, including chikungunya virus (CHIKV) and Ross River virus (RRV), pose significant public health threats because of their ability to cause explosive outbreaks of debilitating arthralgia and myalgia in human populations. Although the host inflammatory response is known to contribute to the pathogenesis of alphavirus-induced arthritis and myositis, the role that Toll-like receptors (TLRs), which are major regulators of host antiviral and inflammatory responses, play in the pathogenesis of alphavirus-induced arthritis and myositis has not been extensively studied. Using a mouse model of RRV-induced myositis/arthritis, we found that myeloid differentiation primary response gene 88 (Myd88)-dependent TLR7 signaling is involved in protection from severe RRV-associated disease. Infections of Myd88- and TLR7-deficient mouse strains with RRV revealed that both Myd88 and TLR7 significantly contributed to protection from RRV-induced mortality, and both mouse strains exhibited more severe tissue damage than wild-type (WT) mice following RRV infection. While viral loads were unchanged in either Myd88 or TLR7 knockout mice compared to WT mice at early times postinfection, both Myd88 and TLR7 knockout mice exhibited higher viral loads than WT mice at late times postinfection. Furthermore, while high levels of RRV-specific antibody were produced in TLR7-deficient mice, this antibody had very little neutralizing activity and had lower affinity than WT antibody. Additionally, TLR7- and Myd88-deficient mice showed defects in germinal center activity, suggesting that TLR7-dependent signaling is critical for the development of protective antibody responses against RRV.
doi:10.1128/JVI.00601-12
PMCID: PMC3457316  PMID: 22837203
8.  Modeling Host Genetic Regulation of Influenza Pathogenesis in the Collaborative Cross 
PLoS Pathogens  2013;9(2):e1003196.
Genetic variation contributes to host responses and outcomes following infection by influenza A virus or other viral infections. Yet narrow windows of disease symptoms and confounding environmental factors have made it difficult to identify polymorphic genes that contribute to differential disease outcomes in human populations. Therefore, to control for these confounding environmental variables in a system that models the levels of genetic diversity found in outbred populations such as humans, we used incipient lines of the highly genetically diverse Collaborative Cross (CC) recombinant inbred (RI) panel (the pre-CC population) to study how genetic variation impacts influenza associated disease across a genetically diverse population. A wide range of variation in influenza disease related phenotypes including virus replication, virus-induced inflammation, and weight loss was observed. Many of the disease associated phenotypes were correlated, with viral replication and virus-induced inflammation being predictors of virus-induced weight loss. Despite these correlations, pre-CC mice with unique and novel disease phenotype combinations were observed. We also identified sets of transcripts (modules) that were correlated with aspects of disease. In order to identify how host genetic polymorphisms contribute to the observed variation in disease, we conducted quantitative trait loci (QTL) mapping. We identified several QTL contributing to specific aspects of the host response including virus-induced weight loss, titer, pulmonary edema, neutrophil recruitment to the airways, and transcriptional expression. Existing whole-genome sequence data was applied to identify high priority candidate genes within QTL regions. A key host response QTL was located at the site of the known anti-influenza Mx1 gene. We sequenced the coding regions of Mx1 in the eight CC founder strains, and identified a novel Mx1 allele that showed reduced ability to inhibit viral replication, while maintaining protection from weight loss.
Author Summary
Host responses to an infectious agent are highly variable across the human population, however, it is not entirely clear how various factors such as pathogen dose, demography, environment and host genetic polymorphisms contribute to variable host responses and infectious outcomes. In this study, a new in vivo experimental model was used that recapitulates many of the genetic characteristics of an outbred population, such as humans. By controlling viral dose, environment and demographic variables, we were able to focus on the role that host genetic variation plays in influenza virus infection. Both the range of disease phenotypes and the combinations of sets of disease phenotypes at 4 days post infection across this population exhibited a large amount of diversity, reminiscent of the variation seen across the human population. Multiple host genome regions were identified that contributed to different aspects of the host response to influenza infection. Taken together, these results emphasize the critical role of host genetics in the response to infectious diseases. Given the breadth of host responses seen within this population, several new models for unique host responses to infection were identified.
doi:10.1371/journal.ppat.1003196
PMCID: PMC3585141  PMID: 23468633
9.  Natural Resistance-associated Macrophage Protein (NRAMP) is a cellular receptor for Sindbis virus in both insect and mammalian hosts 
Cell host & microbe  2011;10(2):97-104.
Summary
Alphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of for this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter Natural Resistance-Associated Macrophage Protein (NRAMP), as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were non-permissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multi-pass membrane proteins for infection.
doi:10.1016/j.chom.2011.06.009
PMCID: PMC3164510  PMID: 21843867
10.  A Double-Inactivated Severe Acute Respiratory Syndrome Coronavirus Vaccine Provides Incomplete Protection in Mice and Induces Increased Eosinophilic Proinflammatory Pulmonary Response upon Challenge▿ 
Journal of Virology  2011;85(23):12201-12215.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is an important emerging virus that is highly pathogenic in aged populations and is maintained with great diversity in zoonotic reservoirs. While a variety of vaccine platforms have shown efficacy in young-animal models and against homologous viral strains, vaccine efficacy has not been thoroughly evaluated using highly pathogenic variants that replicate the acute end stage lung disease phenotypes seen during the human epidemic. Using an adjuvanted and an unadjuvanted double-inactivated SARS-CoV (DIV) vaccine, we demonstrate an eosinophilic immunopathology in aged mice comparable to that seen in mice immunized with the SARS nucleocapsid protein, and poor protection against a nonlethal heterologous challenge. In young and 1-year-old animals, we demonstrate that adjuvanted DIV vaccine provides protection against lethal disease in young animals following homologous and heterologous challenge, although enhanced immune pathology and eosinophilia are evident following heterologous challenge. In the absence of alum, DIV vaccine performed poorly in young animals challenged with lethal homologous or heterologous strains. In contrast, DIV vaccines (both adjuvanted and unadjuvanted) performed poorly in aged-animal models. Importantly, aged animals displayed increased eosinophilic immune pathology in the lungs and were not protected against significant virus replication. These data raise significant concerns regarding DIV vaccine safety and highlight the need for additional studies of the molecular mechanisms governing DIV-induced eosinophilia and vaccine failure, especially in the more vulnerable aged-animal models of human disease.
doi:10.1128/JVI.06048-11
PMCID: PMC3209347  PMID: 21937658
11.  Mannose Binding Lectin Is Required for Alphavirus-Induced Arthritis/Myositis 
PLoS Pathogens  2012;8(3):e1002586.
Mosquito-borne alphaviruses such as chikungunya virus and Ross River virus (RRV) are emerging pathogens capable of causing large-scale epidemics of virus-induced arthritis and myositis. The pathology of RRV-induced disease in both humans and mice is associated with induction of the host inflammatory response within the muscle and joints, and prior studies have demonstrated that the host complement system contributes to development of disease. In this study, we have used a mouse model of RRV-induced disease to identify and characterize which complement activation pathways mediate disease progression after infection, and we have identified the mannose binding lectin (MBL) pathway, but not the classical or alternative complement activation pathways, as essential for development of RRV-induced disease. MBL deposition was enhanced in RRV infected muscle tissue from wild type mice and RRV infected MBL deficient mice exhibited reduced disease, tissue damage, and complement deposition compared to wild-type mice. In contrast, mice deficient for key components of the classical or alternative complement activation pathways still developed severe RRV-induced disease. Further characterization of MBL deficient mice demonstrated that similar to C3−/− mice, viral replication and inflammatory cell recruitment were equivalent to wild type animals, suggesting that RRV-mediated induction of complement dependent immune pathology is largely MBL dependent. Consistent with these findings, human patients diagnosed with RRV disease had elevated serum MBL levels compared to healthy controls, and MBL levels in the serum and synovial fluid correlated with severity of disease. These findings demonstrate a role for MBL in promoting RRV-induced disease in both mice and humans and suggest that the MBL pathway of complement activation may be an effective target for therapeutic intervention for humans suffering from RRV-induced arthritis and myositis.
Author Summary
Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus are transmitted to humans by mosquitoes and cause epidemics of debilitating infectious arthritis and myositis in various areas around the world. Studies in humans and mice indicate that the host inflammatory response is critical for development of RRV-induced arthritis and myositis, and the host complement system, a component of the host inflammatory response, plays an essential role in the development of RRV-induced disease through activation of complement receptor 3 (CR3)-bearing inflammatory cells. Of the three main complement activation pathways, only the lectin pathway activated by mannose binding lectin (MBL) was essential for RRV-induced complement activation, tissue destruction, and disease. Furthermore, we found that levels of MBL were elevated in human patients suffering from RRV-induced polyarthritis and MBL levels correlated with disease severity. Taken together, our data implicates a role for MBL in mediating RRV-induced disease in both humans and mice, and suggests that therapeutic targeting of MBL may be an effective strategy for disease treatment in humans.
doi:10.1371/journal.ppat.1002586
PMCID: PMC3310795  PMID: 22457620
12.  Mutations in nsP1 and PE2 are critical determinants of Ross River virus-induced musculoskeletal inflammatory disease in a mouse model 
Virology  2010;410(1):216-227.
The viral determinants of Alphavirus-induced rheumatic disease have not been elucidated. We identified an RRV strain (DC5692) which, in contrast to the T48 strain, does not induce musculoskeletal inflammation in a mouse model of RRV disease. Substitution of the RRV T48 strain nonstructural protein 1 (nsP1) coding sequence with that from strain DC5692 generated a virus that was attenuated in vivo despite similar viral loads in tissues. In contrast, substitution of the T48 PE2 coding region with the PE2 coding region from DC5692 resulted in attenuation in vivo and reduced viral loads in tissues. In gain of virulence experiments, substitution of the DC5692 strain nsP1 and PE2 coding regions with those from the T48 strain was sufficient to restore full virulence to the DC5692 strain. These findings indicate that determinants in both nsP1 and PE2 have critical and distinct roles in the pathogenesis of RRV-induced musculoskeletal inflammatory disease in mice.
doi:10.1016/j.virol.2010.11.012
PMCID: PMC3017666  PMID: 21131014
Alphavirus; pathogenesis; virulence; inflammation; rheumatic disease
13.  Expression Quantitative Trait Loci for Extreme Host Response to Influenza A in Pre-Collaborative Cross Mice 
G3: Genes|Genomes|Genetics  2012;2(2):213-221.
Outbreaks of influenza occur on a yearly basis, causing a wide range of symptoms across the human population. Although evidence exists that the host response to influenza infection is influenced by genetic differences in the host, this has not been studied in a system with genetic diversity mirroring that of the human population. Here we used mice from 44 influenza-infected pre-Collaborative Cross lines determined to have extreme phenotypes with regard to the host response to influenza A virus infection. Global transcriptome profiling identified 2671 transcripts that were significantly differentially expressed between mice that showed a severe (“high”) and mild (“low”) response to infection. Expression quantitative trait loci mapping was performed on those transcripts that were differentially expressed because of differences in host response phenotype to identify putative regulatory regions potentially controlling their expression. Twenty-one significant expression quantitative trait loci were identified, which allowed direct examination of genes associated with regulation of host response to infection. To perform initial validation of our findings, quantitative polymerase chain reaction was performed in the infected founder strains, and we were able to confirm or partially confirm more than 70% of those tested. In addition, we explored putative causal and reactive (downstream) relationships between the significantly regulated genes and others in the high or low response groups using structural equation modeling. By using systems approaches and a genetically diverse population, we were able to develop a novel framework for identifying the underlying biological subnetworks under host genetic control during influenza virus infection.
doi:10.1534/g3.111.001800
PMCID: PMC3284329  PMID: 22384400
eQTL; influenza; collaborative cross; host response; SEM; Mouse Collaborative Cross; Mouse Genetic Resource
14.  Integrative Deep Sequencing of the Mouse Lung Transcriptome Reveals Differential Expression of Diverse Classes of Small RNAs in Response to Respiratory Virus Infection 
mBio  2011;2(6):e00198-11.
ABSTRACT
We previously reported widespread differential expression of long non-protein-coding RNAs (ncRNAs) in response to virus infection. Here, we expanded the study through small RNA transcriptome sequencing analysis of the host response to both severe acute respiratory syndrome coronavirus (SARS-CoV) and influenza virus infections across four founder mouse strains of the Collaborative Cross, a recombinant inbred mouse resource for mapping complex traits. We observed differential expression of over 200 small RNAs of diverse classes during infection. A majority of identified microRNAs (miRNAs) showed divergent changes in expression across mouse strains with respect to SARS-CoV and influenza virus infections and responded differently to a highly pathogenic reconstructed 1918 virus compared to a minimally pathogenic seasonal influenza virus isolate. Novel insights into miRNA expression changes, including the association with pathogenic outcomes and large differences between in vivo and in vitro experimental systems, were further elucidated by a survey of selected miRNAs across diverse virus infections. The small RNAs identified also included many non-miRNA small RNAs, such as small nucleolar RNAs (snoRNAs), in addition to nonannotated small RNAs. An integrative sequencing analysis of both small RNAs and long transcripts from the same samples showed that the results revealing differential expression of miRNAs during infection were largely due to transcriptional regulation and that the predicted miRNA-mRNA network could modulate global host responses to virus infection in a combinatorial fashion. These findings represent the first integrated sequencing analysis of the response of host small RNAs to virus infection and show that small RNAs are an integrated component of complex networks involved in regulating the host response to infection.
IMPORTANCE
Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, mammalian genomes transcribe many short and long non-protein-coding RNAs (ncRNAs). With the advent of deep-sequencing technologies, systematic transcriptome analysis of the host response, including analysis of ncRNAs of different sizes, is now possible. Using this approach, we recently discovered widespread differential expression of host long (>200 nucleotide [nt]) ncRNAs in response to virus infection. Here, the samples described in the previous report were again used, but we sequenced another fraction of the transcriptome to study very short (about 20 to 30 nt) ncRNAs. We demonstrated that virus infection also altered expression of many short ncRNAs of diverse classes. Putting the results of the two studies together, we show that small RNAs may also play an important role in regulating the host response to virus infection.
doi:10.1128/mBio.00198-11
PMCID: PMC3221602  PMID: 22086488
15.  Successful Vaccination Strategies That Protect Aged Mice from Lethal Challenge from Influenza Virus and Heterologous Severe Acute Respiratory Syndrome Coronavirus ▿  
Journal of Virology  2010;85(1):217-230.
Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP3014) or wild-type VEE glycoproteins (VRP3000) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP3000-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP3014-based vaccines were not. The superior protection for the aged observed with VRP3000-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP3000 vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP3014 platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.
doi:10.1128/JVI.01805-10
PMCID: PMC3014161  PMID: 20980507
16.  A Determinant of Sindbis Virus Neurovirulence Enables Efficient Disruption of Jak/STAT Signaling ▿  
Journal of Virology  2010;84(21):11429-11439.
Previous studies with Venezuelan equine encephalitis virus and Sindbis virus (SINV) indicate that alphaviruses are capable of suppressing the cellular response to type I and type II interferons (IFNs) by disrupting Jak/STAT signaling; however, the relevance of this signaling inhibition toward pathogenesis has not been investigated. The relative abilities of neurovirulent and nonneurovirulent SINV strains to downregulate Jak/STAT signaling were compared to determine whether the ability to inhibit IFN signaling correlates with virulence potential. The adult mouse neurovirulent strain AR86 was found to rapidly and robustly inhibit tyrosine phosphorylation of STAT1 and STAT2 in response to IFN-γ and/or IFN-β. In contrast, the closely related SINV strains Girdwood and TR339, which do not cause detectable disease in adult mice, were relatively inefficient inhibitors of STAT1/2 activation. Decreased STAT activation in AR86-infected cells was associated with decreased activation of the IFN receptor-associated tyrosine kinases Tyk2, Jak1, and Jak2. To identify the viral factor(s) involved, we infected cells with several panels of AR86/Girdwood chimeric viruses. Surprisingly, we found that a single amino acid determinant, threonine at nsP1 position 538, which is required for AR86 virulence, was also required for efficient disruption of STAT1 activation, and this determinant fully restored STAT1 inhibition when it was introduced into the avirulent Girdwood background. These data indicate that a key virulence determinant plays a critical role in downregulating the response to type I and type II IFNs, which suggests that the ability of alphaviruses to inhibit Jak/STAT signaling relates to their in vivo virulence potential.
doi:10.1128/JVI.00577-10
PMCID: PMC2953173  PMID: 20739538
17.  Modulation of type I IFN induction by a virulence determinant within the Alphavirus nsP1 protein 
Virology  2010;399(1):1-10.
Alphaviruses are mosquito-borne viruses that cause serious human and animal diseases. Previous studies demonstrated that a determinant within the nsP1/nsP2 cleavage domain of the virulent Sindbis AR86 virus played a key role in regulating adult mouse virulence without adversely affecting viral replication. Additional characterization of this determinant demonstrated that a virus with the attenuating mutation induced more type I IFN production both in vivo and in vitro. Interestingly, this phenotype was not specific to the Sindbis AR86 virus, as a similar mutation in a distantly related alphavirus, Ross River Virus (RRV), also led to enhanced IFN induction. This effect was independent of virus-induced host shutoff, since IRF-3 phosphorylation, which occurs independently of de novo host transcription/translation, was induced more robustly in cells infected with the mutant viruses. Altogether, these results demonstrate that critical determinants within the nsP1/nsP2 cleavage domain play an important role in regulating alphavirus induced IFN responses.
doi:10.1016/j.virol.2009.12.031
PMCID: PMC2830325  PMID: 20097400
Alphaviruses; Sindbis; Ross River; nsP1 mutants; type I IFN induction
18.  A Protective Role for Complement C3 Protein during Pandemic 2009 H1N1 and H5N1 Influenza A Virus Infection 
PLoS ONE  2011;6(3):e17377.
Highly pathogenic H5N1 influenza infections are associated with enhanced inflammatory and cytokine responses, severe lung damage, and an overall dysregulation of innate immunity. C3, a member of the complement system of serum proteins, is a major component of the innate immune and inflammatory responses. However, the role of this protein in the pathogenesis of H5N1 infection is unknown. Here we demonstrate that H5N1 influenza virus infected mice had increased levels of C5a and C3 activation byproducts as compared to mice infected with either seasonal or pandemic 2009 H1N1 influenza viruses. We hypothesized that the increased complement was associated with the enhanced disease associated with the H5N1 infection. However, studies in knockout mice demonstrated that C3 was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. These studies suggest that although the levels of complement activation may differ depending on the influenza virus subtype, complement is an important host defense mechanism.
doi:10.1371/journal.pone.0017377
PMCID: PMC3052313  PMID: 21408070
19.  Unique Signatures of Long Noncoding RNA Expression in Response to Virus Infection and Altered Innate Immune Signaling 
mBio  2010;1(5):e00206-10.
Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.
IMPORTANCE
Most studies examining the host transcriptional response to infection focus only on protein-coding genes. However, there is growing evidence that thousands of non-protein-coding RNAs (ncRNAs) are transcribed from mammalian genomes. While most attention to the involvement of ncRNAs in virus-host interactions has been on small ncRNAs such as microRNAs, it is becoming apparent that many long ncRNAs (>200 nucleotides [nt]) are also biologically important. These long ncRNAs have been found to have widespread functionality, including chromatin modification and transcriptional regulation and serving as the precursors of small RNAs. With the advent of next-generation sequencing technologies, whole-transcriptome analysis of the host response, including long ncRNAs, is now possible. Using this approach, we demonstrated that virus infection alters the expression of numerous long ncRNAs, suggesting that these RNAs may be a new class of regulatory molecules that play a role in determining the outcome of infection.
doi:10.1128/mBio.00206-10
PMCID: PMC2962437  PMID: 20978541
20.  Modulation of Cellular Tropism and Innate Antiviral Response by Viral Glycans 
Journal of Innate Immunity  2009;1(5):405-412.
Arthropod-borne viruses (arboviruses) are a significant cause of human and animal disease worldwide. Multiple interactions between virus and the host innate immune system ultimately determine the pathogenesis and clinical outcome of the infection. Evidence is rapidly emerging that suggests viral glycans play a key role in viral pathogenesis by regulating host cell tropism and interactions with the host innate immune response. Glycan-mediated interactions are especially important for arboviruses which must adapt to variable glycosylation systems and cellular receptors within both vertebrate and invertebrate hosts. This review focuses on emerging evidence which supports a crucial role for viral glycans in mediating host cell tropism and regulating the innate antiviral response.
doi:10.1159/000226422
PMCID: PMC3214953  PMID: 20375598
Arbovirus; Dendritic cells; Immunity; Glycan; Glycosylation; Lectin; Viral attachment/entry; Virus-host cell interactions
21.  Vaccination with DNA Plasmids Expressing Gn Coupled to C3d or Alphavirus Replicons Expressing Gn Protects Mice against Rift Valley Fever Virus 
Background
Rift Valley fever (RVF) is an arthropod-borne viral zoonosis. Rift Valley fever virus (RVFV) is an important biological threat with the potential to spread to new susceptible areas. In addition, it is a potential biowarfare agent.
Methodology/Principal Findings
We developed two potential vaccines, DNA plasmids and alphavirus replicons, expressing the Gn glycoprotein of RVFV alone or fused to three copies of complement protein, C3d. Each vaccine was administered to mice in an all DNA, all replicon, or a DNA prime/replicon boost strategy and both the humoral and cellular responses were assessed. DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited high titer neutralizing antibodies that were similar to titers elicited by the live-attenuated MP12 virus. Mice vaccinated with an inactivated form of MP12 did elicit high titer antibodies, but these antibodies were unable to neutralize RVFV infection. However, only vaccine strategies incorporating alphavirus replicons elicited cellular responses to Gn. Both vaccines strategies completely prevented weight loss and morbidity and protected against lethal RVFV challenge. Passive transfer of antisera from vaccinated mice into naïve mice showed that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn elicited antibodies that protected mice as well as sera from mice immunized with MP12.
Conclusion/Significance
These results show that both DNA plasmids expressing Gn-C3d and alphavirus replicons expressing Gn administered alone or in a DNA prime/replicon boost strategy are effective RVFV vaccines. These vaccine strategies provide safer alternatives to using live-attenuated RVFV vaccines for human use.
Author Summary
Rift Valley fever virus (RVFV) is an arthropod-borne phlebovirus associated with abortion storms, neonatal mortality in livestock and hemorrhagic fever or fatal encephalitis in a proportion of infected humans. Requirement of multiple booster immunizations to maintain the level of protective immunity with the inactivated vaccines and the ability of live-attenuated vaccines to cause detrimental side-effects are major limitations preventing the widespread use of current vaccines. In this paper, we describe the use of DNA and alphavirus replicon based vaccination approaches to elicit a protective immune response against RVFV. While both vaccines elicited high titer antibodies, DNA vaccination elicited high titer neutralizing antibodies, whereas the replicon vaccine elicited cellular immune responses. Both strategies alone or in combination elicited immune response that completely protected against not only mortality, but also illness. Even though the delivery vectors elicited some protection on their own, they did not prevent severe morbidity. These promising vaccines provide an alternative RVFV vaccine for livestock and humans.
doi:10.1371/journal.pntd.0000725
PMCID: PMC2889828  PMID: 20582312
22.  SARS-CoV Pathogenesis Is Regulated by a STAT1 Dependent but a Type I, II and III Interferon Receptor Independent Mechanism 
PLoS Pathogens  2010;6(4):e1000849.
Severe acute respiratory syndrome coronavirus (SARS-CoV) infection often caused severe end stage lung disease and organizing phase diffuse alveolar damage, especially in the elderly. The virus-host interactions that governed development of these acute end stage lung diseases and death are unknown. To address this question, we evaluated the role of innate immune signaling in protection from human (Urbani) and a recombinant mouse adapted SARS-CoV, designated rMA15. In contrast to most models of viral pathogenesis, infection of type I, type II or type III interferon knockout mice (129 background) with either Urbani or MA15 viruses resulted in clinical disease outcomes, including transient weight loss, denuding bronchiolitis and alveolar inflammation and recovery, identical to that seen in infection of wildtype mice. This suggests that type I, II and III interferon signaling play minor roles in regulating SARS pathogenesis in mouse models. In contrast, infection of STAT1−/− mice resulted in severe disease, high virus titer, extensive pulmonary lesions and 100% mortality by day 9 and 30 post-infection with rMA15 or Urbani viruses, respectively. Non-lethal in BALB/c mice, Urbani SARS-CoV infection in STAT1−/− mice caused disseminated infection involving the liver, spleen and other tissues after day 9. These findings demonstrated that SARS-CoV pathogenesis is regulated by a STAT1 dependent but type I, II and III interferon receptor independent, mechanism. In contrast to a well documented role in innate immunity, we propose that STAT1 also protects mice via its role as an antagonist of unrestrained cell proliferation.
Author Summary
The SARS coronavirus is a highly pathogenic respiratory virus that caused the first epidemic of the 21st century. During the epidemic ∼10% of those infected died and the elderly were particularly vulnerable. Severe cases developed acute lung injury with pulmonary fibrosis and Acute Respiratory Distress Syndrome (ARDS). Little is known about the molecular mechanisms governing its virus pathogenesis and high lethality. Using a mouse model of infection with the epidemic strain of SARS-CoV (Urbani) as well as a recombinant mouse adapted strain of SARS-CoV (rMA15), we showed that a protein normally associated with the innate immune response, STAT1, plays an important role in the development of severe end stage lung injury. However, the lack of a normal innate immune type I, type II and type III interferon response did not enhance virus pathogenesis. Our work suggests that STAT1 may play a key role in development of acute lung injury and other chronic lung pathology, most likely by affecting cell proliferation and wound repair pathways.
doi:10.1371/journal.ppat.1000849
PMCID: PMC2851658  PMID: 20386712
23.  Venezuelan Equine Encephalitis Virus Disrupts STAT1 Signaling by Distinct Mechanisms Independent of Host Shutoff▿  
Journal of Virology  2009;83(20):10571-10581.
Venezuelan equine encephalitis virus (VEEV) is an important human and veterinary pathogen causing sporadic epizootic outbreaks of potentially fatal encephalitis. The type I interferon (IFN) system plays a central role in controlling VEEV and other alphavirus infections, and IFN evasion is likely an important determinant of whether these viruses disseminate and cause disease within their hosts. Alphaviruses are thought to limit the induction of type I IFNs and IFN-stimulated genes by shutting off host cell macromolecular synthesis, which in the case of VEEV is partially mediated by the viral capsid protein. However, more specific strategies by which alphaviruses inhibit type I IFN signaling have not been characterized. Analyses of cells infected with VEEV and VEEV replicon particles (VRP) demonstrate that viral infection rapidly disrupts tyrosine phosphorylation and nuclear translocation of the transcription factor STAT1 in response to both IFN-β and IFN-γ. This effect was independent of host shutoff and expression of viral capsid, suggesting that VEEV uses novel mechanisms to interfere with type I and type II IFN signaling. Furthermore, at times when STAT1 activation was efficiently inhibited, VRP infection did not limit tyrosine phosphorylation of Jak1, Tyk2, or STAT2 after IFN-β treatment but did inhibit Jak1 and Jak2 activation in response to IFN-γ, suggesting that VEEV interferes with STAT1 activation by the type I and II receptor complexes through distinct mechanisms. Identification of the viral requirements for this novel STAT1 inhibition will further our understanding of alphavirus molecular pathogenesis and may provide insights into effective alphavirus-based vaccine design.
doi:10.1128/JVI.01041-09
PMCID: PMC2753124  PMID: 19656875
24.  Ross River Virus Envelope Glycans Contribute to Type I Interferon Production in Myeloid Dendritic Cells▿  
Journal of Virology  2008;82(24):12374-12383.
Alphaviruses are mosquito-transmitted viruses that cause significant human disease, and understanding how these pathogens successfully transition from the mosquito vector to the vertebrate host is an important area of research. Previous studies demonstrated that mosquito and mammalian-cell-derived alphaviruses differentially induce type I interferons (alpha/beta interferon [IFN-α/β]) in myeloid dendritic cells (mDCs), where the mosquito cell-derived virus is a poor inducer of IFN-α/β compared to the mammalian-cell-derived virus. Furthermore, the reduced IFN-α/β induction by the mosquito cell-derived virus is attributed to differential N-linked glycosylation (29). To further evaluate the role of viral envelope glycans in regulating the IFN-α/β response, studies were performed to assess whether the mosquito cell-derived virus actively inhibits IFN-α/β induction or is simply a poor inducer of IFN-α/β. Coinfection studies using mammalian- and mosquito cell-derived Ross River virus (mam-RRV and mos-RRV, respectively) indicated that mos-RRV was unable to suppress IFN-α/β induction by mam-RRV in mDC cultures. Additionally, a panel of mutant viruses lacking either individual or multiple N-linked glycosylation sites was used to demonstrate that N-linked glycans were essential for high-level IFN-α/β induction by the mammalian-cell-derived virus. These results suggest that the failure of the mosquito cell-derived virus to induce IFN-α/β is due to a lack of complex carbohydrates on the virion rather than the active suppression of the DC antiviral response.
doi:10.1128/JVI.00985-08
PMCID: PMC2593332  PMID: 18922878
25.  Complement Receptor 3 Promotes Severe Ross River Virus-Induced Disease▿  
Journal of Virology  2008;82(22):11263-11272.
Alphaviruses such as Ross River virus (RRV) and chikungunya virus are mosquito-transmitted viruses that cause explosive epidemics of debilitating arthritis and myositis affecting millions of humans worldwide. Previous studies using a mouse model of RRV-induced disease demonstrated that viral infection results in a severe inflammatory arthritis and myositis and that complement component 3 (C3) contributes to the destructive phase of the inflammatory disease but not the recruitment of cellular infiltrates to the sites of RRV-induced inflammation. Here, we demonstrate that mice deficient in complement receptor 3 (CR3) (CD11b−/−), a signaling receptor activated by multiple ligands including the C3 cleavage fragment iC3b, develop less-severe disease signs and decreased tissue destruction compared to RRV-infected wild-type mice. CR3 deficiency had no effect on viral replication, nor did it diminish the magnitude, kinetics, and composition of the cellular infiltrates at the sites of inflammation. However, the genetic absence of CR3 diminished the expression of specific proinflammatory and cytotoxic effectors, including S100A9/S100A8 and interleukin-6, within the inflamed tissues, suggesting that CR3-dependent signaling at the sites of inflammation contributes to tissue damage and severe disease.
doi:10.1128/JVI.01352-08
PMCID: PMC2573283  PMID: 18787004

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